Optical lens system and method for microfluidic devices

ABSTRACT

An apparatus for imaging one or more selected fluorescence indications from a microfluidic device. The apparatus includes an imaging path coupled to least one chamber in at least one microfluidic device. The imaging path provides for transmission of one or more fluorescent emission signals derived from one or more samples in the at least one chamber of the at least one microfluidic device. The chamber has a chamber size, the chamber size being characterized by an actual spatial dimension normal to the imaging path. The apparatus also includes an optical lens system coupled to the imaging path. The optical lens system is adapted to transmit the one or more fluorescent signals associated with the chamber.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 14/547,442filed Nov. 19, 2104, which is a continuation of U.S. Ser. No. 13/937,340filed Jul. 9, 2013 (now U.S. Pat. No. 8,926,905), which is acontinuation of U.S. Ser. No. 13/253,703 filed Oct. 5, 2011 (now U.S.Pat. No. 8,512,640), which is a continuation of U.S. Ser. No. 12/862,621filed Aug. 24, 2010 (now U.S. Pat. No. 8,048,378), which is acontinuation of U.S. Ser. No. 12/538,641 filed Aug. 10, 2009 (now U.S.Pat. No. 7,906,072), which is a division of U.S. Ser. No. 11/953,538filed Dec. 10, 2007 (now U.S. Pat. No. 7,588,672), which is acontinuation of U.S. Ser. No. 11/148,157 filed Jun. 7, 2005 (now U.S.Pat. No. 7,307,802), which claims priority to U.S. ProvisionalApplication No. 60/578,106 filed Jun. 7, 2004—the disclosures of whichare all incorporated herein by reference in their entirety for allpurposes.

BACKGROUND OF THE INVENTION

The present invention relates generally to microfluidic techniques. Inparticular, the invention provides a method and system for imaging oneor more entities in a chamber of a microfluidic device (e.g., suspendedin a volume of fluid). More particularly, the present method and systemfor imaging uses indications from a fluorescence signal associated withthe one or more entities in the microfluidic device. Merely by way ofexample, the techniques for microfluidic methods and systems are appliedusing fluorescent, chemiluminescent, and bioluminescent readers coupledto the microfluidic device, but it would be recognized that theinvention has a much broader range of applicability.

Concerted efforts to develop and manufacture microfluidic systems toperform various chemical and biochemical analyses and syntheses haveoccurred. Such systems have been developed for preparative andanalytical applications. A goal to make such micro-sized devices arisesfrom significant benefits achieved from miniaturization of conventionalmacro scale analyses and syntheses, which are often cumbersome and lessefficient. A substantial reduction in time, lower costs, and moreefficient space allocation are achieved as benefits using thesemicrofluidic systems. Additional benefits may include a reduction inhuman operator involvement with automated systems using thesemicrofluidic devices. Automated systems also decrease operator errorsand other operator type limitations. Microfluidic devices have beenproposed for use in a variety of applications, including, for instance,capillary electrophoresis, gas chromatography and cell separations.

Microfluidic devices adapted to conduct nucleic acid amplificationprocesses are potentially useful in a wide variety of applications. Forexample, such devices could be used to determine the presence or absenceof a particular target nucleic acid in a sample, as an analytical tool.Examples of utilizing microfluidic device as an analytical tool include:

-   -   testing for the presence of particular pathogens (e.g., viruses,        bacteria or fungi);    -   identification processes (e.g., paternity and forensic        applications);    -   detecting and characterizing specific nucleic acids associated        with particular diseases or genetic disorders;    -   detecting gene expression profiles/sequences associated with        particular drug behavior (e.g. for pharmacogenetics, i.e.        choosing drugs which are compatible/especially efficacious        for/not hazardous with specific genetic profiles); and    -   conducting genotyping analyses and gene expression analyses        (e.g., differential gene expression studies).

Alternatively, the devices can be used in a preparative fashion toamplify nucleic acids, producing an amplified product at sufficientlevels needed for further analysis. Examples of these analysis processesinclude sequencing of the amplified product, cell-typing, DNAfingerprinting, and the like. Amplified products can also be used invarious genetic engineering applications. These genetic engineeringapplications include (but are not limited to) the production of adesired protein product, accomplished by insertion of the amplifiedproduct into a vector that is then used to transform cells into thedesired protein product.

Despite these potential applications, imaging systems (also referred toas readers) adapted to collect and process imaging data, for example,fluorescence data, from such microfluidic devices have variousshortcomings. Some conventional readers operate in a scanning mode, inwhich a laser beam is raster scanned over the microfluidic device. Inother such systems, the device or both the laser and the device aretranslated. These scanners collect fluorescence data from the reactionchambers present in the microfluidic device in a sequential mannerassociated with the raster scanning of the laser source/device. Otherconventional scanners operate in a stitching mode, sequentially imagingsmall areas, for example, areas less than 1 mm² in size, and stitchingthese small images together to form an image of the microfluidic deviceunder test.

Both scanning and stitching systems have shortcomings. For example, bothtypes of systems operate at a relatively low system frequency, which isproportional to the area imaged as a function of time. Conventionalsystems operate at frequencies on the order of 1-20 cm² per minute. Forsome interesting assays, such as protein calorimetry and nucleic acidamplification, system frequencies greater than about 1-20 cm² per minuteare generally required to image the fluorescent processes occurring inthe reaction vessels of the microfluidic device. Conventional scanningand stitching systems are not able to meet these performance goals. Inaddition to slowing system throughput, these scanning and stitchingsystem can limit the potential for utilizing certain assays, e.g.,performance of real-time PCR.

Therefore, there is a need in the art for improved methods and systemsfor imaging one or more entities suspended in a volume of fluid in achamber of a microfluidic device.

SUMMARY OF THE INVENTION

According to the present invention, techniques for microfluidic systemsare provided. In particular, the invention provides a method and systemfor imaging one or more entities suspended in a volume of fluid in achamber of a microfluidic device. More particularly, the present methodand system for imaging uses indications from a fluorescence signalassociated with the one or more entities in the microfluidic device.Merely by way of example, the techniques for microfluidic methods andsystems are applied using fluorescent, chemiluminescent, andbioluminescent readers coupled to the microfluidic device, but it wouldbe recognized that the invention has a much broader range ofapplicability.

In a specific embodiment, the present invention provides an apparatusfor imaging one or more selected fluorescence indications from amicrofluidic device. The apparatus includes an imaging path coupled toleast one chamber in at least one microfluidic device. The imaging pathprovides for transmission of one or more fluorescent emission signalsderived from one or more samples in the at least one chamber of the atleast one microfluidic device. The chamber has a chamber size, thechamber size being characterized by an actual spatial dimension normalto the imaging path. The apparatus also includes an optical lens systemcoupled to the imaging path. The optical lens system is adapted totransmit the one or more fluorescent signals associated with thechamber.

In another specific embodiment, a method of imaging one or more selectedfluorescence indications from at least one chamber of a microfluidicdevice is provided. The method includes transmitting one or morefluorescent emission signals derived from one or more samples in the atleast one chamber of at least one microfluidic device along an imagingpath coupled to the at least one chamber. The at least one chamber has achamber size, the chamber size being characterized by an actual spatialdimension normal to the imaging path. The method also includestransmitting the one or more fluorescent emission signals associatedwith the chamber through an optical lens system coupled to the imagingpath. The optical lens system is adapted to reduce a size of the actualspatial dimension to a determined level.

In yet another specific embodiment of the present invention, a systemfor imaging one or more indications from one or more chambers of amicrofluidic device is provided. The system includes an optical path,the optical path being capable of transmitting one or more images of aportion of a spatial region of a microfluidic device from the portion ofthe spatial region of the microfluidic device. In an embodiment, theportion of the spatial region of the microfluidic device ischaracterized by a first dimension. The system also includes a firstlens system coupled to a first portion of the optical path. The firstlens system is characterized by a first optical characteristic. Thesystem further includes a second lens system coupled to a second portionof the optical path. The second lens system is characterized by a secondoptical characteristic. The system additionally includes a detectordevice coupled to a third portion of the optical path. The detectordevice is operable to capture the one or more images of the portion ofthe spatial region. Moreover, the detector is adapted to capture the oneor more images. The one or more images have a determined size at thedetector device of about the first dimension or less.

In an alternative embodiment, a method for imaging one or moreindications from one or more chambers of a microfluidic device. Themethod includes transmitting one or more images of a portion of aspatial region of a microfluidic device from the portion of the spatialregion of the microfluidic device along an optical path. The portion ofthe spatial region of the microfluidic device is characterized by afirst dimension. The method also includes coupling a first lens systemto a first portion of the optical path. The first lens system ischaracterized by a first optical characteristic. The method additionallyincludes coupling a second lens system to a second portion of theoptical path. The second lens system is characterized by a secondoptical characteristic. Moreover, the method includes capturing the oneor more images of the portion of the spatial region using a detectordevice. The detector device is coupled to a third portion of the opticalpath and the one or more images have a determined size at the detectordevice of about the first dimension or less.

In another alternative embodiment, a method of imaging microfluidicdevices is provided the method includes capturing an image of a spatialregion associated with at least a determined number of chambers of amicrofluidic device using an image detection spatial region during atime frame of less than one minute. In a specific embodiment, thecapturing of the image of the spatial region is substantially free froma stitching and/or scanning process.

In yet another alternative embodiment, an apparatus for imaging one ormore selected fluorescence indications from a microfluidic device isprovided. The apparatus includes an imaging path coupled to least onechamber in at least one microfluidic device. The imaging path providesfor transmission of one or more fluorescent emission signals derivedfrom one or more samples in the at least one chamber of the at least onemicrofluidic device. The apparatus also includes an optical filterdevice coupled to a first spatial portion of the imaging path providedfor transmission of the one or more emission signals. The optical filterdevice is adapted to transmit a selected spectral bandwidth from the oneor more fluorescent emission signals and is adapted to process one ormore chromatic aberrations associated with the one or more fluorescentemission signals to a determined level.

In a particular embodiment, a method of analyzing processes inelastomeric microfluidic devices is provided. The method includescapturing an image of at least 96 chambers in a time period of less thanone minute. In an embodiment, each of the chambers in the at least 96chambers is in fluidic isolation from any of the other chambers in theat least 96 chambers. The method also includes processing the image.

In another particular embodiment, an apparatus for imaging anmicrofluidic device comprising a plurality of processing sites isprovided. The plurality of processing sites contain at least one sampleselected from M samples and at least one reagent selected from Nreagents. The apparatus includes an illumination system coupled to themicrofluidic device and adapted to illuminate the microfluidic devicewith electromagnetic radiation. The apparatus also includes an imagingsystem coupled to the microfluidic device and adapted to receiveelectromagnetic radiation emitted from the plurality of processingsites. The apparatus additionally includes a detector coupled to theimaging system.

In yet another particular embodiment, an optical imaging system isprovided. The optical imaging system includes a computer and an opticalillumination system adapted to illuminate an elastomeric microfluidicarray device including at least 1,536 reaction chambers in fluidicisolation. The elastomeric microfluidic array device includes anelastomeric block formed from a plurality of layers. At least one layerof the plurality of layers has at least one recess formed therein. Therecess has at least one deflectable membrane integral to the layer withthe recess. The optical imaging system also includes an opticaldetection system.

In yet another alternative particular embodiment, a method of imagingone or more selected fluorescence indications from a plurality ofchambers in a microfluidic device is provided. The method includestransmitting one or more fluorescent emission signals along an imagingpath, the one or more fluorescent emission signals derived from one ormore samples in at least one of the plurality of chambers in themicrofluidic device. The method also includes selectively transmitting asubset of the one or more fluorescent emission signals along the imagingpath utilizing an optical filter device adapted to pass fluorescentemission signals within a predetermined spectral bandwidth and adaptedto process one or more chromatic aberrations associated with the one ormore fluorescent emission signals to a determined level.

In a specific embodiment, the method further includes reading a portionof the subset of the one or more fluorescent emission signals at adetector, capturing the one or more fluorescent emission signals derivedfrom the one or more samples in at least one of the plurality ofchambers in the microfluidic device, irradiating at least 96 chambers inthe at least one microfluidic device, wherein each of the chambers in agroup of more than 48 chambers is in fluidic isolation from any of theother chambers in the group of more than 48 chambers, and maintainingthe at least one microfluidic device at a predetermined temperature at adetermined time, wherein the predetermined temperature at thepredetermined time is a portion of a multi-step thermocycling profile.In another specific embodiment, processing the one or more chromaticaberrations associated with the one or more fluorescent emission signalsincludes reducing the one or more chromatic aberrations to apredetermined level. The predetermined level is characterized by apredetermined shift in a focal point associated with a first raycharacterized by a first color and a second ray characterized by asecond color. In yet another specific embodiment, the optical filterdevice includes a plurality of zero-power doublets and a plurality ofspectral filters.

In an embodiment according to the present invention, the use of anoptical imaging system to collect information related to an microfluidicdevice is provided. In a specific embodiment, the use of the imagingsystem includes a microfluidic device comprising greater than 63chambers. In another specific embodiment, the use of the imaging systemincludes a microfluidic device that has an elastomeric microfluidicdevice. In an alternative embodiment, the use of the imaging systemincludes a microfluidic device comprising greater than 95 chambers. Inanother alternative embodiment, the use of the imaging system includes amicrofluidic device comprising greater than 383 chambers. In yet anotheralternative embodiment, the use of the imaging system includes amicrofluidic device comprising greater than 511 chambers. In anadditional embodiment, the use of the imaging system includes amicrofluidic device comprising greater than or equal to 2,304 chambers.In another additional embodiment, the use of the imaging system includesa microfluidic device comprising greater than or equal to 9,216chambers. In yet another additional embodiment, the use of the imagingsystem includes a microfluidic device comprising greater than or equalto 100,000 chambers.

According to another embodiment of the present invention, the use of anoptical imaging system to collect information related to an microfluidicdevice is provided. The use of the optical system includes anelastomeric microfluidic device adapted to perform proteincrystallization processes in a particular embodiment. In anotherembodiment, the use of imaging system includes an elastomericmicrofluidic device that is adapted to perform fluorescence processes.In yet another embodiment, the use of the imaging system includes anelastomeric microfluidic device that is adapted to perform reactions ata selected temperature or range of temperatures over time. In anotherspecific embodiment, the optical imaging system is used to collectinformation related to PCR reactions. In yet another specificembodiment, the use of the imaging system includes a microfluidic devicecomprising closed reaction chambers in microfluidic isolation. In otherembodiments, the use of the imaging system includes a thermal controllerthat is coupled to the microfluidic device.

In an alternative embodiment, the use of an optical imaging system toimage a microfluidic device is provided. In this alternative embodiment,the optical imaging system has a plurality of doublets adapted to reducea value of chromatic aberration to a predetermined level. In anembodiment, the optical imaging system is characterized by an NA greaterthan 0.23. In another embodiment, the optical imaging system has an NAgreater than or equal to 0.36. In yet another embodiment, the opticalimaging system has an NA greater than 0.2. In a specific embodiment, theoptical imaging system comprises a first spectral filter coupled to afirst doublet and a second spectral filter coupled to a second doublet.Moreover, in an additional embodiment, the first doublet reduces a valueof chromatic aberration at a first wavelength passed by the firstspectral filter to a first predetermined level and the second doubletreduces a value of chromatic aberration at a second wavelength passed bythe second spectral filter to a second predetermined level. In someembodiments, the first doublet and the second doublet are zero-powerdoublets. In other embodiments, the first spectral filter, the firstdoublet, the second spectral filter, and the second doublet are arrangedas a filter wheel.

In another embodiment of the present invention, the use of afluorescence imaging system to acquire a plurality of images, includingone or more fluorescent emission signals, from an microfluidic device isprovided. The use of the fluorescence imaging system includes amicrofluidic device that has greater than 63 reaction chambers influidic isolation from other reaction chambers. In an embodiment, theuse of the fluorescence imaging system includes a microfluidic devicethat has an elastomeric microfluidic device. In another embodiment, theuse of the fluorescence imaging system includes a plurality of reactionchambers that are characterized by a volume of less than 10 nl. In yetanother embodiment, the use of the fluorescence imaging system includesan elastomeric microfluidic device that is characterized by an arraydensity greater than or equal to 100 reaction chambers per cm². In analternative embodiment, the fluorescence imaging system includes anoptical filter device, the optical filter device being adapted to pass aselected spectral bandwidth from the one or more fluorescent emissionsignals and being adapted to process one or more chromatic aberrationsassociated with the one or more fluorescent emission signals to adetermined level.

In an embodiment of the present invention, an optical system forinterrogating samples in a microfluidic system is provided. In theoptical system provided by this embodiment, the detector consists of anarray (e.g. a CCD) equal to or larger in dimensions than themicrofluidic system which is imaged onto it. In another embodiment, thedetector is optically coupled to the microfluidic device using at leastone lens. In yet another embodiment, the detector is not in conformalcontact with the microfluidic device. In a specific embodiment, thedetector is in conformal contact with the microfluidic device. Inanother specific embodiment, chromatic aberration correction systems asdiscussed throughout the specification are utilized in the opticaltrain.

In another embodiment, an optical system for interrogating amicrofluidic device is provided. The optical system has a magnificationM≧NA_(o)/NA_(det), where NA_(o) is the object/sample-side NA andNA_(det) is the maximum NA allowable onto the detector before losses dueto reflection occurring at the detector face pass a threshold value. Ina specific embodiment, the detector is a CCD and NA_(det)=0.36. Inanother embodiment, the detector is a CCD and 0.23≦NA_(det)≦0.5.

In an additional embodiment, an optical system for interrogating samplesin a microfluidic system is provided. The detector comprises an array(e.g. a CCD) equal to M times the dimensions of the area of themicrofluidic device containing reaction chambers. In an embodiment, thearea of the microfluidic device includes the sample chambers to beinterrogated. In this embodiment, M=NA_(o)/NA_(det)+/−10%, where NA_(o)is the maximum NA acceptable as determined by depth-of-focusconsiderations related to the acceptable crosstalk between reactionchambers, their XY spacing and size, and their extent along the Z axis,and NA_(det) is such that 40% of incident light is lost at the detectorsurface due to reflections, vignetting, and the like. In a specificembodiment, the NA_(det) is such that ≦30% of incident light is lost atthe detector surface due to reflections, vignetting, and the like. Inanother specific embodiment, the NA_(det) is such that ≦20% of incidentlight is lost at the detector surface due to reflections, vignetting,and the like. In yet another specific embodiment, the NA_(det) is suchthat ≦10% of incident light is lost at the detector surface due toreflections, vignetting, and the like. In an alternative embodiment, Mis ≧1. In another alternative embodiment, M=1.

Numerous benefits are achieved using the present invention overconventional techniques. Some embodiments provide optical imagingsystems to generate and detect fluorescence from a microfluidic device.Additionally, embodiments of the present invention provide opticalsystems that reduce chromatic aberration through the use of zero-powerachromatic doublets coupled to spectral filters.

The capability of performing a number of applications on a singleplatform are provided by embodiments of the present invention. Forexample genomics applications including gene expression and genotypingare performed using embodiments of the present invention. Moreover,Digital Isolation and Detection (DID) applications are made possible. Asan example, applications related to cancer detection as well as singlecell macro molecule detection and quantification are provided.Furthermore, proteomics applications including protein-ligand bindingand an immunoassay processes are provided through embodiments of thepresent invention.

Additionally, depending upon the embodiments of the present invention,certain benefits and/or advantages may be achieved to overcome certainlimitations associated with other techniques. The following has beendetermined based upon calculations:

Some scanning systems have a desired resolution of 10 um. In thesesystems, there are a total of 3000×3000 (=approximately 10⁶⁷) “spots”for which fluorescence is desirably quantified. For example, toaccomplish this in 10 seconds, the residence time at each spot is ˜1microsecond. Accordingly, a linescan (3000 points) would be complete in3 ms. To get equivalent signal-to-noise to that achieved usingembodiments of the present invention, the light intensity should be ˜10⁶times as bright in the spot as would be achieved by an imaging systemaccording to the present invention acquiring a signal over a period of 1second. To raster a spot across 3 cm in 3 ms, 1000 times per second, isoften mechanically challenging. In addition, collection of the emissionsignal with high efficiency is desired. This generally requires either alarge field optic or a high NA flying head (e.g., moving at >=10 m/s).In this case, the data collected at the lower right hand corner of adevice will be 10 seconds older than the data collected in the upperleft hand corner of the device, which is undesirable in someapplications.

Some stitching systems acquire multiple images and stitch them togetherto form a composite image. In one system, in order to stitch together animage from multiple 3 mm×3 mm images, 100 images are utilized. The totaltime used to acquire these individual images would be 100 ms. Assumingthat approximately half the time is spent moving and half the time isspent acquiring an image, the image time will be ˜50 ms. This systemwould generally require a light intensity ˜20 times as intense asutilized in embodiments of the present invention acquiring a signal overa period of 1 second, and/or a higher-NA objective. In addition, thesesystems generally need a fast stage—capable of moving 3 mm and coming toa complete stop in 50 ms. Additionally, these systems generally requirecareful calibration of both motion and light intensity to eliminateimage artifacts from stitching. The data collected at the lower righthand corner of the device will be 10 seconds older than the datacollected in the upper left hand corner, which is undesirable in someapplications.

Some embodiments of the present invention provide imaging systems inwhich the area of the microfluidic device is imaged all at once. In anembodiment in which 10 seconds per image is available, several secondsof integration time are available. Synchronization problems are reducedor eliminated as data is collected from all the pixels at the same time.Moreover, embodiments of the present invention generally illuminate theentire microfluidic device (˜31 mm×31 mm area) at once and utilize lenssystems with high NAs and a large field of view. As described throughoutthe present specification, embodiments of the present invention providemethods and systems that are less complex and more powerful thanconventional systems. Depending upon the embodiment, one or more ofthese limitations may be overcome by the present methods and systemsaccording to embodiments of the present invention. Further details ofthe present invention may be found throughout the present specificationand more particularly below. Depending upon the embodiment, one or moreof these benefits may exist. These and other benefits have beendescribed throughout the present specification and more particularlybelow.

Various additional objects, features and advantages of the presentinvention can be more fully appreciated with reference to the detaileddescription and accompanying drawings that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a simplified schematic diagram illustrating an opticalimaging system according to an embodiment of the present invention;

FIG. 1B depicts an overview of an exemplary imaging system according toan alternative embodiment of the present invention;

FIG. 1C is a simplified schematic diagram illustrating a thermal controldevice according to a embodiment of the present invention;

FIGS. 2A-2C are simplified schematic diagrams illustrating a lensassembly according to an embodiment of the present invention;

FIG. 3 is a photograph of fluorescent emission centered at a firstwavelength produced by a reaction occurring in a number of reactionchambers present in a microfluidic device according to an embodiment ofthe present invention;

FIG. 4 is a photograph of fluorescent emission centered at a secondwavelength produced by a reaction occurring in a number of reactionchambers present in a microfluidic device according to an embodiment ofthe present invention;

FIGS. 5-7 are spot diagrams for selected wavelengths produced using anembodiment of the present invention;

FIG. 8 is an illumination diagram illustrating relative uniformity as afunction of position produced using an embodiment of the presentinvention;

FIGS. 9-11 are ensquared energy diagrams for several embodimentsaccording to the present invention;

FIG. 12 is a diagram illustrating field curvature and distortion for anoptical system provided according to an embodiment of the presentinvention;

FIG. 13 is a diagram illustrating double wavelength versus focusproduced by systems according to an embodiment of the present invention;

FIGS. 14-16 are spot diagrams for selected wavelengths produced using anembodiment of the present invention;

FIGS. 17-19 are spot diagrams for selected wavelengths produced using anembodiment of the present invention;

FIGS. 20-22 are spot diagrams for selected wavelengths produced using anembodiment of the present invention;

FIGS. 23-25 are spot diagrams for selected wavelengths produced using anembodiment of the present invention; and

FIGS. 26-28 are ensquared energy diagrams for several embodimentsaccording to the present invention.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

According to the present invention, techniques for microfluidic systemsare provided. In particular, the invention provides a method and systemfor imaging one or more entities suspended in a volume of fluid in achamber of a microfluidic device. More particularly, the present methodand system for imaging uses indications from a fluorescence signalassociated with the one or more entities in the microfluidic device.Merely by way of example, the techniques for microfluidic methods andsystems are applied using fluorescent, chemiluminescent, andbioluminescent readers coupled to the microfluidic device, but it wouldbe recognized that the invention has a much broader range ofapplicability.

In the present application, references are made to certain types of“reaction” chambers in a microfluidic device. In general, these“reaction chambers” include processing sites, processing chambers,and/or reaction sites, any combination of these, and the like. Thesechambers may be closed, partially closed, open, partially open, sealed,or combinations thereof, including any temporary or transient conditionsinvolving any of these states, and the like. In some embodiments, thechambers are sealed, capable of being sealed, closeable, isolated,capable of being isolated, and combinations thereof, and any combinationor single condition of any temporary or transient conditions involvingany of these states, and the like. Therefore, use of the term reactionchamber is not intended to limit the present invention, but to includethese other structures. Additionally, the term chamber is not intendedto limit the present invention, but should be used in its ordinarymeaning, unless specific features associated with the chamber have beenrecited. Of course, there can be other variations, modifications, andalternatives.

Moreover, through the present application, references are made tofluorescent indications from a microfluidic device. Included within thescope of the present invention are not only fluorescent indications, butluminescent indications, including chemiluminescent, electroluminescent,electrochemiluminescent, and phospholuminescent, bioluminescent, andother luminescent processes, or any other processing involving any othertype of indications that may be detected using a detection device. Aswill be evident to one of skill in the art, methods and systems operablein the detection and analysis of these fluorescent and luminescentindications are transferable from one indication to another.Additionally, although some embodiments of the present invention utilizespectral filters as optical elements, this is not required by thepresent invention. Some fluorescent and luminescent applications do notutilize spectral filters in the optical excitation path, the opticalemission path, or both. As described herein, other embodiments utilizespectral filters. One of skill in the art will appreciate thedifferences associated with particular applications.

In some embodiments, a variety of devices and methods for conductingmicrofluidic analyses are utilized herein, including devices that can beutilized to conduct thermal cycling reactions such as nucleic acidamplification reactions. The devices differ from conventionalmicrofluidic devices in that they include elastomeric components; insome instances, much or all of the device is composed of elastomericmaterial. For example, amplification reactions can be linearamplifications, (amplifications with a single primer), as well asexponential amplifications (i.e., amplifications conducted with aforward and reverse primer set).

The methods and systems provided by some embodiments of the presentinvention utilize blind channel type devices in performing nucleic acidamplification reactions. In these devices, the reagents that aretypically deposited within the reaction sites are those reagentsnecessary to perform the desired type of amplification reaction. Usuallythis means that some or all of the following are deposited: primers,polymerase, nucleotides, metal ions, buffer, and cofactors, for example.The sample introduced into the reaction site in such cases is thenucleic acid template. Alternatively, however, the template can bedeposited and the amplification reagents flowed into the reaction sites.As discussed in more detail throughout the present specification, when amatrix device is utilized to conduct an amplification reaction, samplescontaining nucleic acid template are flowed through the vertical flowchannels and the amplification reagents through the horizontal flowchannels or vice versa.

PCR is perhaps the best known amplification technique. The devicesutilized in embodiments of the present invention are not limited toconducting PCR amplifications. Other types of amplification reactionsthat can be conducted include, but are not limited to, (i) ligase chainreaction (LCR) (see Wu and Wallace, Genomics 4:560 (1989) and Landegrenet al., Science 241:1077 (1988)); (ii) transcription amplification (seeKwoh et al., Proc. Natl. Acad. Sci. USA 86:1173 (1989)); (iii)self-sustained sequence replication (see Guatelli et al., Proc. Nat.Acad. Sci. USA, 87:1874 (1990)); and (iv) nucleic acid based sequenceamplification (NASBA) (see, Sooknanan, R. and Malek, L., BioTechnology13: 563-65 (1995)). Each of the foregoing references are incorporatedherein by reference in their entirety for all purposes.

Moreover, certain devices are designed to conduct thermal cyclingreactions (e.g., PCR) with devices that include one or more elastomericvalves to regulate solution flow through the device. Thus, methods forconducting amplification reactions with devices of this design are alsoprovided.

Amplification products (amplicons) can be detected and distinguished(whether isolated in a reaction chamber or at any subsequent time) usingroutine methods for detecting nucleic acids. Amplicons comprisingdouble-stranded DNA can be detected using intercalation dyes such asSYBR™, Pico Green (Molecular Probes, Inc., Eugene, Oreg.), ethidiumbromide and the like (see Zhu et al., 1994, Anal. Chem. 66:1941-48)and/or gel electrophoresis. More often, sequence-specific detectionmethods are used (i.e., amplicons are detected based on their nucleotidesequence). Examples of detection methods include hybridization to arraysof immobilized oligo or polynucleotides, and use of differentiallylabeled molecular beacons or other “fluorescence resonance energytransfer” (FRET)-based detection systems. FRET-based detection is apreferred method for detection according to some embodiments of thepresent invention. In FRET-based assays a change in fluorescence from adonor (reporter) and/or acceptor (quencher) fluorophore in adonor/acceptor fluorophore pair is detected. The donor and acceptorfluorophore pair are selected such that the emission spectrum of thedonor overlaps the excitation spectrum of the acceptor. Thus, when thepair of fluorophores are brought within sufficiently close proximity toone another, energy transfer from the donor to the acceptor can occurand can be detected. A variety of assays are known including, forexample and not limitation, template extension reactions, quantitativeRT-PCR, Molecular Beacons, and Invader assays, these are describedbriefly below.

FRET and template extension reactions utilize a primer labeled with onemember of a donor/acceptor pair and a nucleotide labeled with the othermember of the donor/acceptor pair. Prior to incorporation of the labelednucleotide into the primer during an template-dependent extensionreaction, the donor and acceptor are spaced far enough apart that energytransfer cannot occur. However, if the labeled nucleotide isincorporated into the primer and the spacing is sufficiently close, thenenergy transfer occurs and can be detected. These methods areparticularly useful in conducting single base pair extension reactionsin the detection of single nucleotide polymorphisms and are described inU.S. Pat. No. 5,945,283 and PCT Publication WO 97/22719. The reactionscan optionally be thermocycled to increase signal using the temperaturecontrol methods and apparatus described throughout the presentspecification.

A variety of so-called “real time amplification” methods or “real timequantitative PCR” methods can also be used to determine the quantity ofa target nucleic acid present in a sample by measuring the amount ofamplification product formed during or after the amplification processitself. Fluorogenic nuclease assays are one specific example of a realtime quantitation method which can be used successfully with the devicesdescribed herein. This method of monitoring the formation ofamplification product involves the continuous measurement of PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe—anapproach frequently referred to in the literature as the “TaqMan”method. See, for example, U.S. Pat. No. 5,723,591.

With molecular beacons, a change in conformation of the probe as ithybridizes to a complementary region of the amplified product results inthe formation of a detectable signal. The probe itself includes twosections: one section at the 5′ end and the other section at the 3′ end.These sections flank the section of the probe that anneals to the probebinding site and are complementary to one another. One end section istypically attached to a reporter dye and the other end section isusually attached to a quencher dye. In solution, the two end sectionscan hybridize with each other to form a hairpin loop. In thisconformation, the reporter and quencher dye are in sufficiently closeproximity that fluorescence from the reporter dye is effectivelyquenched by the quencher dye. Hybridized probe, in contrast, results ina linearized conformation in which the extent of quenching is decreased.Thus, by monitoring emission changes for the two dyes, it is possible toindirectly monitor the formation of amplification product. Probes ofthis type and methods of their use are described further, for example,by Piatek et al., 1998, Nat. Biotechnol. 16:359-63; Tyagi, and Kramer,1996, Nat. Biotechnology 14:303-308; and Tyagi, et al., 1998, Nat.Biotechnol. 16:49-53 (1998).

The Scorpion detection method is described, for example, by Thelwell etal. 2000, Nucleic Acids Research, 28:3752-3761 and Solinas et al., 2001,“Duplex Scorpion primers in SNP analysis and FRET applications” NucleicAcids Research 29:20. Scorpion primers are fluorogenic PCR primers witha probe element attached at the 5′-end via a PCR stopper. They are usedin real-time amplicon-specific detection of PCR products in homogeneoussolution. Two different formats are possible, the ‘stem-loop’ format andthe ‘duplex’ format. In both cases the probing mechanism isintramolecular. The basic elements of Scorpions in all formats are: (i)a PCR primer; (ii) a PCR stopper to prevent PCR read-through of theprobe element; (iii) a specific probe sequence; and (iv) a fluorescencedetection system containing at least one fluorophore and quencher. AfterPCR extension of the Scorpion primer, the resultant amplicon contains asequence that is complementary to the probe, which is renderedsingle-stranded during the denaturation stage of each PCR cycle. Oncooling, the probe is free to bind to this complementary sequence,producing an increase in fluorescence, as the quencher is no longer inthe vicinity of the fluorophore. The PCR stopper prevents undesirableread-through of the probe by Taq DNA polymerase.

Invader assays (Third Wave Technologies, Madison, Wis.) are usedparticularly for SNP genotyping and utilize an oligonucleotide,designated the signal probe, that is complementary to the target nucleicacid (DNA or RNA) or polymorphism site. A second oligonucleotide,designated the Invader Oligo, contains the same 5′ nucleotide sequence,but the 3′ nucleotide sequence contains a nucleotide polymorphism. TheInvader Oligo interferes with the binding of the signal probe to thetarget nucleic acid such that the 5′ end of the signal probe forms a“flap” at the nucleotide containing the polymorphism. This complex isrecognized by a structure specific endonuclease, called the Cleavaseenzyme. Cleavase cleaves the 5′ flap of the nucleotides. The releasedflap binds with a third probe bearing FRET labels, thereby forminganother duplex structure recognized by the Cleavase enzyme. This timethe Cleavase enzyme cleaves a fluorophore away from a quencher andproduces a fluorescent signal. For SNP genotyping, the signal probe willbe designed to hybridize with either the reference (wild type) allele orthe variant (mutant) allele. Unlike PCR, there is a linear amplificationof signal with no amplification of the nucleic acid. Further detailssufficient to guide one of ordinary skill in the art are provided by,for example, Neri, B. P., et al., Advances in Nucleic Acid and ProteinAnalysis 3826:117-125, 2000) and U.S. Pat. No. 6,706,471.

A variety of multiplex amplification systems can be used in conjunctionwith the present invention. In one type, several different targets canbe detected simultaneously by using multiple differently labeled probeseach of which is designed to hybridize only to a particular target.Since each probe has a different label, binding to each target to bedetected based on the fluorescence signals. By judicious choice of thedifferent labels that are utilized, analyses can be conducted in whichthe different labels are excited and/or detected at differentwavelengths in a single reaction. See, e.g., Fluorescence Spectroscopy(Pesce et al., Eds.) Marcel Dekker, New York, (1971); White et al.,Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York,(1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules,2nd ed., Academic Press, New York, (1971); Griffiths, Colour andConstitution of Organic Molecules, Academic Press, New York, (1976);Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and Haugland,Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Eugene (1992).

Many diseases linked to genome modifications, either of the hostorganism or of infectious organisms, are the consequence of a change ina small number of nucleotides, frequently involving a change in a singlenucleotide. Such single nucleotide changes are referred to as singlenucleotide polymorphisms or simply SNPs, and the site at which the SNPoccurs is typically referred to as a polymorphic site. The devicesdescribed herein can be utilized to determine the identify of anucleotide present at such polymorphic sites. As an extension of thiscapability, the devices can be utilized in genotyping analyses.Genotyping involves the determination of whether a diploid organism(i.e., an organism with two copies of each gene) contains two copies ofa reference allele (a reference-type homozygote), one copy each of thereference and a variant allele (i.e., a heterozygote), or contains twocopies of the variant allele (i.e., a variant-type homozygote). Whenconducting a genotyping analysis, the methods of the invention can beutilized to interrogate a single variant site. However, as describedfurther below in the section on multiplexing, the methods can also beused to determine the genotype of an individual in many different DNAloci, either on the same gene, different genes or combinations thereof.

Devices to be utilized for conducting genotyping analyses are designedto utilize reaction sites of appropriate size to ensure from astatistical standpoint that a copy of each of the two alleles for adiploid subject are present in the reaction site at a workable DNAconcentrations. Otherwise, an analysis could yield results suggestingthat a heterozygote is a homozygote simply because a copy of the secondallele is not present at the reaction site. Table 1 below indicates thenumber of copies of the genome present in a 1 nl reaction volume atvarious exemplary DNA concentrations that can be utilized with thedevices described herein.

TABLE 1 Number of genome copies present in a 1 nanoliter volume at theindicated DNA concentration. Volume (nanoliter) [DNA] (μg/μL) N 1 0.33100 1 0.10 32 1 0.05 16 1 0.01 3 1 0.003 1

As a general matter, due to stochastic proportioning of the sample, thecopy number present before an amplification reaction is commenceddetermines the likely error in the measurement. Genotyping analysesusing certain devices are typically conducted with samples having a DNAconcentration of approximately 0.10 μg/μL, although the currentinventors have run successful TaqMan reactions at concentrations inwhich there is a single genome per reaction site.

Genotyping analyses can be conducted using a variety of differentapproaches. In these methods, it is generally sufficient to obtain a“yes” or “no” result, i.e., detection need only be able to answer thequestion whether a given allele is present. Thus, analyses can beconducted only with the primers or nucleotides necessary to detect thepresence of one allele potentially at a polymorphic site. However, moretypically, primers and nucleotides to detect the presence of each allelepotentially at the polymorphic site are included.

Single Base Pair Extension (SBPE) reactions are one techniquespecifically developed for conducting genotyping analyses. Although anumber of SPBE assays have been developed, the general approach is quitesimilar. Typically, these assays involve hybridizing a primer that iscomplementary to a target nucleic acid such that the 3′ end of theprimer is immediately 5′ of the variant site or is adjacent thereto.Extension is conducted in the presence of one or more labelednon-extendible nucleotides that are complementary to the nucleotide(s)that occupy the variant site and a polymerase. The non-extendiblenucleotide is a nucleotide analog that prevents further extension by thepolymerase once incorporated into the primer. If the addednon-extendible nucleotide(s) is(are) complementary to the nucleotide atthe variant site, then a labeled non-extendible nucleotide isincorporated onto the 3′ end of the primer to generate a labeledextension product. Hence, extended primers provide an indication ofwhich nucleotide is present at the variant site of a target nucleicacid. Such methods and related methods are discussed, for example, inU.S. Pat. Nos. 5,846,710; 6,004,744; 5,888,819; 5,856,092; and5,710,028; and in WO 92/16657.

Genotyping analyses can also be conducted using quantitative PCRmethods. In this case, differentially labeled probes complementary toeach of the allelic forms are included as reagents, together withprimers, nucleotides and polymerase. However, reactions can be conductedwith only a single probe, although this can create ambiguity as towhether lack of signal is due to absence of a particular allele orsimply a failed reaction. For the typical biallelic case in which twoalleles are possible for a polymorphic site, two differentially labeledprobes, each perfectly complementary to one of the alleles are usuallyincluded in the reagent mixture, together with amplification primers,nucleotides and polymerase. Sample containing the target DNA isintroduced into the reaction site. If the allele to which a probe iscomplementary is present in the target DNA, then amplification occurs,thereby resulting in a detectable signal as described in the detectionabove. Based upon which of the differential signal is obtained, theidentity of the nucleotide at the polymorphic site can be determined. Ifboth signals are detected, then both alleles are present. Thermocyclingduring the reaction is performed as described in the temperature controlsection supra.

Gene expression analysis involves determining the level at which one ormore genes is expressed in a particular cell. The determination can bequalitative, but generally is quantitative. In a differential geneexpression analysis, the levels of the gene(s) in one cell (e.g., a testcell) are compared to the expression levels of the same genes in anothercell (control cell). A wide variety of such comparisons can be made.Examples include, but are not limited to, a comparison between healthyand diseased cells, between cells from an individual treated with onedrug and cells from another untreated individual, between cells exposedto a particular toxicant and cells not exposed, and so on. Genes whoseexpression levels vary between the test and control cells can serve asmarkers and/or targets for therapy. For example, if a certain group ofgenes is found to be up-regulated in diseased cells rather than healthycells, such genes can serve as markers of the disease and canpotentially be utilized as the basis for diagnostic tests. These genescould also be targets. A strategy for treating the disease might includeprocedures that result in a reduction of expression of the up-regulatedgenes.

The design of the microfluidic devices utilized in embodiments of thepresent invention is helpful in facilitating a variety of geneexpression analyses. Because the devices contain a large number ofreaction sites, a large number of genes and/or samples can be tested atthe same time. Using the blind flow channel devices, for instance, theexpression levels of hundreds or thousands of genes can be determined atthe same time. The devices also facilitate differential gene expressionanalyses. With the matrix design, for example, a sample obtained from ahealthy cell can be tested in one flow channel, with a sample from adiseased cell run in an immediately adjacent channel. This featureenhances the ease of detection and the accuracy of the results becausethe two samples are run on the same device at the same time and underthe same conditions.

A variety of matrix or array-based devices are also utilized accordingto embodiments of the present invention. Certain of these devicesinclude: (i) a first plurality of flow channels formed in an elastomericsubstrate, (ii) a second plurality of flow channels formed in theelastomeric substrate that intersect the first plurality of flowchannels to define an array of reaction sites, (iii) a plurality ofisolation valves disposed within the first and second plurality of flowchannels that can be actuated to isolate solution within each of thereaction sites from solution at other reaction sites, and (iv) aplurality of guard channels overlaying one or more of the flow channelsand/or one or more of the reaction sites to prevent evaporation ofsolution therefrom. The foregoing devices can be utilized to conduct anumber of different types of reactions, including those involvingtemperature regulation (e.g., thermocycling of nucleic acid analyses).

Some of the microfluidic devices utilize a design typically referred toherein as “blind channel” or “blind fill” and are characterized in partby having a plurality of blind channels, which are flow channels havinga dead end or isolated end such that solution can only enter and exitthe blind channel at one end (i.e., there is not a separate inlet andoutlet for the blind channel). These devices require only a single valvefor each blind channel to isolate a region of the blind channel to forman isolated reaction site. During manufacture of this type of device,one or more reagents for conducting an analysis are deposited at thereaction sites, thereby resulting in a significant reduction in thenumber of input and outputs. Additionally, the blind channels areconnected to an interconnected network of channels such that all thereaction sites can be filled from a single, or limited number, of sampleinputs. Because of the reduction in complexity in inputs and outputs andthe use of only a single valve to isolate each reaction site, the spaceavailable for reaction sites is increased. Thus, the features of thesedevices means that each device can include a large number of reactionsites (e.g., up to tens of thousands) and can achieve high reaction sitedensities (e.g., over 1,000-4,000 reaction sites/cm²). Individually andcollectively, these features also directly translate into a significantreduction in the size of these devices compared to traditionalmicrofluidic devices.

Other microfluidic devices that are disclosed herein utilize a matrixdesign. In general, microfluidic devices of this type utilize aplurality of intersecting horizontal and vertical flow channels todefine an array of reaction sites at the points of intersection. Thus,devices of this design also have an array or reaction sites; however,there is a larger number of sample inputs and corresponding outputs toaccommodate the larger number of samples with this design. A valvesystem referred to as a switchable flow array architecture enablessolution be flowed selectively through just the horizontal or flowchannels, thus allowing for switchable isolation of various flowchannels in the matrix. Hence, whereas the blind channel devices aredesigned to conduct a large number of analyses under differentconditions with a limited number of samples, the matrix devices areconstructed to analyze a large number of samples under a limited numberof conditions. Still other devices are hybrids of these two generaldesign types.

Other microfluidic devices are massively partitioning devices (DID) suchas described in PCT publication WO 2004/089810, U.S. patent applicationSer. No. 10/819,088 filed Apr. 5, 2004 (now U.S. Pat. No. 7,691,333),copending commonly assigned patent application No. 60/687,010 filed Jun.2, 2006 entitled “Analysis using microfluidic partitioning devices,”each of which are incorporated by reference in their entirety for allpurposes. Using massively partitioning devices, a sample can bepartitioned into a multitude of isolated reaction chambers, andreactions carried out simultaneously in each chamber.

The microfluidic devices that are described herein are furthercharacterized in part by utilizing various components such as flowchannels, control channels, valves and/or pumps fabricated fromelastomeric materials. In some instances, essentially the entire deviceis made of elastomeric materials. Consequently, such devices differsignificantly in form and function from the majority of conventionalmicrofluidic devices that are formed from plastics or silicon-basedmaterials. The number of reaction chambers provided varies according toembodiments of the present invention.

The design of the devices enables them to be utilized in combinationwith a number of different heating systems. Thus, the devices are usefulin conducting diverse analyses that require temperature control.Additionally, those microfluidic devices adapted for use in heatingapplications can incorporate a further design feature to minimizeevaporation of sample from the reaction sites. Devices of this type ingeneral include a number of guard channels and/or reservoirs or chambersformed within the elastomeric device through which water can be flowedto increase the water vapor pressure within the elastomeric materialfrom which the device is formed, thereby reducing evaporation of samplematerial from the reaction sites.

In another embodiment, a temperature cycling device may be used tocontrol the temperature of the microfluidic devices. Preferably, themicrofluidic device would be adapted to make thermal contact with themicrofluidic device. Where the microfluidic device is supported by asubstrate material, such as a glass slide or the bottom of a carrierplate, such as a plastic carrier, a window may be formed in a region ofthe carrier or slide such that the microfluidic device, preferably adevice having an elastomeric block, may directly contact theheating/cooling block of the temperature cycling device. In a preferredembodiment, the heating/cooling block has grooves therein incommunication with a vacuum source for applying a suction force to themicrofluidic device, preferably a portion adjacent to where thereactions are taking place. Alternatively, a rigid thermally conductiveplate may be bonded to the microfluidic device that then mates with theheating and cooling block for efficient thermal conduction resulting.

The array format of certain of the devices means the devices can achievehigh throughput. Collectively, the high throughput and temperaturecontrol capabilities make the devices useful for performing largenumbers of nucleic acid amplifications (e.g., polymerase chain reaction(PCR)). Such reactions will be discussed at length herein asillustrative of the utility of the devices, especially of their use inany reaction requiring temperature control. However, it should beunderstood that the devices are not limited to these particularapplications. The devices can be utilized in a wide variety of othertypes of analyses or reactions. Examples include analyses ofprotein-ligand interactions and interactions between cells and variouscompounds. Further examples are provided throughout the presentspecification.

The microfluidic devices disclosed herein are typically constructed atleast in part from elastomeric materials and constructed by single andmultilayer soft lithography (MSL) techniques and/or sacrificial-layerencapsulation methods (see, e.g., Unger et al. (2000) Science288:113-116, and PCT Publication WO 01/01025, both of which areincorporated by reference herein in their entirety for all purposes).Utilizing such methods, microfluidic devices can be designed in whichsolution flow through flow channels of the device is controlled, atleast in part, with one or more control channels that are separated fromthe flow channel by an elastomeric membrane or segment. This membrane orsegment can be deflected into or retracted from the flow channel withwhich a control channel is associated by applying an actuation force tothe control channels. By controlling the degree to which the membrane isdeflected into or retracted out from the flow channel, solution flow canbe slowed or entirely blocked through the flow channel. Usingcombinations of control and flow channels of this type, one can preparea variety of different types of valves and pumps for regulating solutionflow as described in extensive detail in Unger et al. (2000) Science288:113-116, and PCT Publication WO 02/43615 and WO 01/01025.

The devices provided herein incorporate such pumps and/or valves toisolate selectively a reaction site at which reagents are allowed toreact. Alternatively, devices without pumps and/or valves are utilizedthat use pressure driven flow or polymerization processes to closeappropriate channels and thereby selectively isolate reaction sites. Thereaction sites can be located at any of a number of different locationswithin the device. For example, in some matrix-type devices, thereaction site is located at the intersection of a set of flow channels.In blind channel devices, the reaction site is located at the end of theblind channel.

If the device is to be utilized in temperature control reactions (e.g.,thermocycling reactions), then, as described in greater detail infra,the elastomeric device is typically fixed to a support (e.g., a glassslide). The resulting structure can then be placed on a temperaturecontrol plate, for example, to control the temperature at the variousreaction sites. In the case of thermocycling reactions, the device canbe placed on any of a number of thermocycling plates.

Because the devices are made of elastomeric materials that arerelatively optically transparent, reactions can be readily monitoredusing a variety of different detection systems at essentially anylocation on the microfluidic device. Most typically, however, detectionoccurs at the reaction site itself (e.g., within a region that includesan intersection of flow channels or at the blind end of a flow channel).The fact that the device is manufactured from substantially transparentmaterials also means that certain detection systems can be utilized withthe current devices that are not usable with traditional silicon-basedmicrofluidic devices. Detection can be achieved using detectors that areincorporated into the device or that are separate from the device butaligned with the region of the device to be detected.

Devices utilizing the matrix design generally have a plurality ofvertical and horizontal flow channels that intersect to form an array ofjunctions. Because a different sample and reagent (or set of reagents)can be introduced into each of the flow channels, a large number ofsamples can be tested against a relatively large number of reactionconditions in a high throughput format. Thus, for example, if adifferent sample is introduced into each of M different vertical flowchannels and a different reagent (or set of reagents) is introduced intoeach of N horizontal flow channels, then M×N different reactions can beconducted at the same time. Typically, matrix devices include valvesthat allow for switchable isolation of the vertical and horizontal flowchannels. Said differently, the valves are positioned to allow selectiveflow just through the vertical flow channels or just through thehorizontal flow channels. Because devices of this type allow flexibilitywith respect to the selection of the type and number of samples, as wellas the number and type of reagents, these devices are well-suited forconducting analyses in which one wants to screen a large number ofsamples against a relatively large number of reaction conditions. Thematrix devices can optionally incorporate guard channels to help preventevaporation of sample and reactants.

Some high-density matrix designs utilize fluid communication viasbetween layers of the microfluidic device to weave control lines andfluid lines through the device. For example, by having a fluid line ineach layer of a two layer elastomeric block, higher density reactioncell arrangements are possible. As will be evident to one of skill inthe art, multi-layer devices allow fluid lines to cross over or undereach other without being in fluid communication. For example, in aparticular design, a reagent fluid channel in a first layer is connectedto a reagent fluid channel in a second layer through a via, while thesecond layer also has sample channels therein, the sample channels andthe reagent channels terminating in sample and reagent chambers,respectively. The sample and reagent chambers are in fluid communicationwith each other through an interface channel that has an interface valveassociated therewith to control fluid communication between each of thechambers of a reaction cell. In use, the interface is first closed, thenreagent is introduced into the reagent channel from the reagent inletand sample is introduced into the sample channel through the sampleinlet. Containment valves are then closed to isolate each reaction cellfrom other reaction cells. Once the reaction cells are isolated, theinterface valve is opened to cause the sample chamber and the reagentchamber to be in fluid communication with each other so that a desiredreaction may take place. One of ordinary skill in the art wouldrecognize many variations, modifications, and alternatives.

Accordingly, a particular design for a microfluidic device provides fora microfluidic device adapted to react M number of different sampleswith N number of different reagents comprising: a plurality of reactioncells, each reaction cell comprising a sample chamber and a reagentchamber, the sample chamber and the reagent chamber being in fluidcommunication through an interface channel having an interface valveassociated therewith for controlling fluid communication between thesample chamber and the reagent chamber; a plurality of sample inletseach in fluid communication with the sample chambers; a plurality ofreagent inlets each in fluid communication with the reagent chambers;wherein one of the sample inlets or reagent inlets is in fluidcommunication with one of the sample chambers or one of the reagentchambers, respectively, through a via. Certain embodiments includehaving the reaction cells be formed within an elastomeric block formedfrom a plurality of layers bonded together and the interface valve is adeflectable membrane; having the sample inlets be in communication withthe sample chamber through a sample channel and the reagent inlet influid communication with the reagent chamber through a reagent channel,a portion of the sample channel and a portion of the reagent channelbeing oriented about parallel to each other and each having acontainment valve associated therewith for controlling fluidcommunication therethrough; having the valve associated with the samplechannel and the valve associated with the reagent channel in operablecommunication with each other through a common containment controlchannel; having the containment common control channel located along aline about normal to one of the sample channel or the reagent channel.

The microfluidic devices utilized in embodiments of the presentinvention may be further integrated into the carrier devices describedin co-pending and commonly owned U.S. Patent Application No. 60/557,715by Unger filed on Mar. 29, 2004, which is incorporated herein for allpurposes. The carrier of Unger provides on-board continuous fluidpressure to maintain valve closure away from a source of fluid pressure,e.g., house air pressure. Unger further provides for an automated systemfor charging and actuating the valves of the present invention asdescribed therein. An another preferred embodiment, the automated systemfor charging accumulators and actuating valves employs a device having aplaten that mates against one or more surfaces of the microfluidicdevice, wherein the platen has at least two or more ports in fluidcommunication with a controlled vacuum or pressure source, and mayinclude mechanical portions for manipulating portions of themicrofluidic device, for example, but not limited to, check valves.

Another device utilized in embodiments of the present invention providesa carrier used as a substrate for stabilizing an elastomeric block.Preferably the carrier has one or more of the following features; a wellor reservoir in fluid communication with the elastomeric block throughat least one channel formed in or with the carrier; an accumulator influid communication with the elastomeric block through at least onechannel formed in or with the carrier; and, a fluid port in fluidcommunication with the elastomeric block, wherein the fluid port ispreferably accessible to an automated source of vacuum or pressure, suchas the automated system described above, wherein the automated sourcefurther comprises a platen having a port that mates with the fluid portto form an isolated fluid connection between the automated system forapplying fluid pressure or vacuum to the elastomeric block. In devicesutilized in certain embodiments, the automated source can also makefluid communication with one or more accumulators associated with thecarrier for charging and discharging pressure maintained in anaccumulator. In certain embodiments, the carrier may further comprise aregion located in an area of the carrier that contacts the microfluidicdevice, wherein the region is made from a material different fromanother portion of the carrier, the material of the region beingselected for improved thermal conduction and distribution propertiesthat are different from the other portion of the carrier. Preferredmaterials for improved thermal conduction and distribution include, butare not limited to silicon, preferably silicon that is highly polished,such as the type of silicon available in the semiconductor field as apolished wafer or a portion cut from the wafer, e.g., chip.

As described more fully below, embodiments of the present inventionutilize a thermal source, for example, but not limited to a PCRthermocycler, which may have been modified from its originalmanufactured state. Generally the thermal source has a thermallyregulated portion that can mate with a portion of the carrier,preferably the thermal conduction and distribution portion of thecarrier, for providing thermal control to the elastomeric block throughthe thermal conduction and distribution portion of the carrier. In apreferred embodiment, thermal contact is improved by applying a sourceof vacuum to a one or more channels formed within the thermallyregulated portion of the thermal source, wherein the channels are formedto contact a surface of the thermal conduction and distribution portionof the carrier to apply suction to and maintain the position of thethermal conduction and distribution portion of the carrier. In apreferred embodiment, the thermal conduction and distribution portion ofthe carrier is not in physical contact with the remainder of thecarrier, but is associated with the remainder of the carrier and theelastomeric block by affixing the thermal conduction and distributionportion to the elastomeric block only and leaving a gap surrounding theedges of the thermal conduction and distribution portion to reduceparasitic thermal effects caused by the carrier. It should be understoodthat in many aspects of the invention described herein, the preferredelastomeric block could be replaced with any of the known microfluidicdevices in the art not described herein, for example devices producedsuch as the GeneChip® by Affymetrix® of Santa Clara, Calif., USA, or byCaliper of Mountain View, Calif., USA. U.S. patents issued to Soane,Parce, Fodor, Wilding, Ekstrom, Quake, or Unger, describe microfluidicor mesoscale fluidic devices that can be substituted for the elastomericblock of the present invention to take advantage of the thermaladvantages and improvements, e.g., suction positioning, reducingparasitic thermal transfer to other regions of the fluidic device, whichare described above in the context of using an elastomeric block.

Utilizing systems and methods provided according to embodiments of thepresent invention, throughput increases are provided over 384 wellsystems. As an example, throughput increases of a factor of 4, 6, 12,and 24 and greater are provided in some embodiments. These throughputincreases are provided while reducing the logistical friction ofoperations. Moreover the systems and methods of embodiments of thepresent invention enable multiple assays for multiple samples. Forexample, in a specific embodiment 96 samples and 96 assays are utilizedto provide a total of 9,216 data points. In a particular example, the 96assays are components of a TaqMan 5′ Nuclease Assay.

Furthermore, embodiments of the present invention provide reducedreaction volumes. In embodiments of the present invention, reactionvolumes ranging from 10 picoliters to 100 nanoliters are utilized. Insome embodiments, reaction volumes greater than 100 nanoliters areutilized. Merely by way of example, in an embodiment, the methods andsystems of the present invention are utilized with reaction volumes of10 picoliters, 50 picoliters, 100 picoliters, 250 picoliters, 500picoliters, and 1 nanoliter. In alternative embodiments, reactionvolumes of 2 nanoliters, 5 nanoliters, 10 nanoliters, 20 nanoliters, 30nanoliters, 40 nanoliters, 50 nanoliters, 75 nanoliters, and 100nanoliters are utilized.

Depending on the geometry of the particular microfluidic device and thesize of the microfluidic device and the arrangement of the fluidcommunication paths and processing site, embodiments of the presentinvention provide for a range of processing site (or reaction chamber)densities. In some embodiments, the methods and systems of the presentinvention are utilized with chamber densities ranging from about 100chambers per cm² to about 1 million chambers per cm². Merely by way ofexample, microfluidic devices with chamber densities of 250, 1,000,2,500, 10,000, 25,000, 100,000, and 250,000 chambers per cm² areutilized according to embodiments of the present invention. In someembodiments, chamber densities in excess of 1,000,000 chambers per cm²are utilized, although this is not required by the present invention.

Operating microfluidic devices with such small reaction volumes reducesreagent usage as well as sample usage. Moreover, some embodiments of thepresent invention provide methods and systems adapted to performreal-time detection, when used in combination with real-timequantitative PCR. Utilizing these systems and methods, six orders oflinear dynamic range are provided for some applications as well asquantitative resolution high enough to allow for the detection ofsub-nanoMolar fluorophore concentrations in 10 nanoliter volumes. One ofordinary skill in the art would recognize many variations,modifications, and alternatives.

Methods conducted with certain blind channel type devices involveproviding a microfluidic device that comprises a flow channel formedwithin an elastomeric material; and a plurality of blind flow channelsin fluid communication with the flow channel, with an end region of eachblind flow channel defining a reaction site. At least one reagent isintroduced into each of the reaction sites, and then a reaction isdetected at one or more of the reaction sites. The method can optionallyinclude heating the at least one reagent within the reaction site. Thus,for example, a method can involve introducing the components for anucleic acid amplification reaction and then thermocycling thecomponents to form amplified product. As more fully described below, anoptical imaging system adapted to characterize reactions occurring incertain microfluidic devices is provided according to embodiments of thepresent invention.

As illustrated in FIG. 1A, optical imaging systems provided according tosome embodiments of the present invention include fluorescence imagingsystems coupled to thermal control modules. Such systems are adapted tocollect data from microfluidic chips with N×M geometries. In someembodiments, N is equal to M. For example, embodiments of the presentinvention utilize microfluidic devices with 48×48 reaction chambers,96×96 reaction chambers, and other geometries. In a particularembodiment, 96 samples and 96 reagents are utilized in a microfluidicdevice with a 96×96 reaction chamber geometry. As will be evident to oneof skill in the art, the methods and systems provided according toembodiments of the present invention enable one platform to performmultiple applications.

FIG. 1A is a simplified schematic diagram illustrating an opticalimaging system according to an embodiment of the present invention. Asillustrated in FIG. 1A, an optical source 242 is provided according toembodiments of the present invention. As will be described more fullybelow, in some embodiments of the present invention, light from opticalsource 242 is utilized to induce fluorescence in a sample. In otherembodiments, chemiluminescence is utilized as a indicator. Depending onthe embodiment, system components will be added, removed, or used, aswill be evident to one of skill in the art. In various embodiments,optical sources including light emitting diodes (LEDs), lasers, arclamps, incandescent lamps, and the like are utilized. These sources maybe polychromatic or monochromatic. In a particular embodiment, theoptical source is characterized by a first spectral bandwidth. In aspecific embodiment, the optical source is a white light sourceproducing optical radiation over a spectral range from about 400 nm toabout 700 nm. Merely by way of example, a Lambda LS 300W Xenon Arc lamp,available from Sutter Instruments of Novato, Calif. is utilized as anoptical source is some embodiments of the present invention. As will beevident to one of skill in the art, other optical sources characterizedby larger or smaller spectral bandwidths are capable of being utilizedin alternative embodiments.

Excitation filter wheel 244 is illustrated in FIG. 1A. In someembodiments, for example, those in which the optical source ispolychromatic, the excitation filter wheel 244 is utilized to spectrallyfilter the light emitted by the optical source 242. Of course, multiplefilters could also be used. As an example, in an embodiment, theexcitation filter wheel provides a number of spectral filters eachadapted to pass a predetermined wavelength range as appropriate forexciting specific fluorescence from a sample. As illustrated in FIG. 1A,the excitation filter wheel 244 is coupled to computer 270, providingfor computer control of the filters. In a particular embodiment, theexcitation filter wheel provides a number of spectral filters:

Filter 1: A filter with a center wavelength of 485 nm and a spectralbandwidth of 20 nm;

Filter 2: A filter with a center wavelength of 530 nm and a spectralbandwidth of 20 nm; and

Filter 3: A filter with a center wavelength of 580 nm and a spectralbandwidth of 20 nm.

As will be evident to one of skill in the art, embodiments of thepresent invention are not limited to these particular spectral filters,but will utilize spectral filters adapted for fluorescence processes forparticular samples. Moreover, although the previous discussion relatedto the use of a filter wheel, this is not required by the presentinvention. In alternative embodiments, spectral filters are provided ingeometries other than a wheel. For example, spectral filters that dropinto a filter holder, electro-optic filters, filters placed into theoptical path by actuators, and the like are included according toembodiments of the present invention. Moreover, in other embodiments,the optical source is a tunable laser adapted to emit radiation atpredetermined wavelengths suitable for excitation of fluorescence. Oneof ordinary skill in the art would recognize many variations,modifications, and alternatives.

As illustrated in FIG. 1A, excitation shutter 246 is provided accordingto embodiments of the present invention. The excitation shutter isoperated under control of a computer 270 in some embodiments, toblock/pass the optical signal generated by the optical source 242 andspectrally filtered by the excitation filter wheel 244. Depending on theapplication, the excitation source is blocked while samples are insertedand removed from the system as well as for calibration operations. Insome embodiments, the excitation shutter is not utilized, for example,in embodiments utilizing laser sources, which provide alternative meansto extinguish the optical source.

When the excitation shutter is operated in an open position, the opticalexcitation signal passes through a fiber bundle 248 and is directed soas to impinge on a microfluidic device 205 provided in chip carrier to aseven. Other embodiments of the present invention utilize quartz lightguides, liquid light guides, other scrambling systems, and the like toincrease illumination homogeneity. As illustrated in FIG. 1A, theexcitation optical signal is directed, through reflection by opticalilluminator 250, refraction, or combinations thereof, to impinge on asurface of the microfluidic device 205. As illustrated in FIG. 1A,illumination of the microfluidic device is via optical illuminator 250.In other embodiments illumination maybe coupled to the microfluidicdevice obliquely from one or more sides of device, via a ring light, orvia a portion of the collection optical train (the optical path betweenthe microfluidic device and the detector 260.

In some embodiments, the illumination of the microfluidic device withlight produced by the excitation source is provided over atwo-dimensional area of the sample. In these embodiments, a large fieldof view is provided, which enables the performance of fluorescenceapplications that involve imaging of time resolved chemical processesand reactions. As an example, fluorescent imaging of protein calorimetryand nucleic acid amplification processes are time resolved processesthat benefit from embodiments of the present invention. In some of theseprocesses, simultaneously excitation of the fluorescent samples providedin a number of reaction chambers and simultaneous collection of thefluorescent signals produced by the reactions occurring in the number ofreaction chambers is desirable. In other processes, for instance,fluorescence lifetime imaging, a brief excitation pulse is followed bydetection (and analysis) of the fluorescent signal as it decays in timefrom an initial level. One of ordinary skill in the art would recognizemany variations, modifications, and alternatives.

As an example, nucleic acid amplification processes typically includethe target DNA, a thermostable DNA polymerase, two oligonucleotideprimers, deoxynucleotide triphosphates (dNTPs), a reaction buffer, andmagnesium. Once assembled, the reaction is placed in a thermal cycler,an instrument that subjects the reaction to a series of differenttemperatures for varying amounts of time. This series of temperature andtime adjustments is referred to as one cycle of amplification. Eachcycle theoretically doubles the amount of targeted sequence (amplicon)in the reaction. Ten cycles theoretically multiply the amplicon by afactor of about one thousand; 20 cycles, by a factor of more than amillion in a matter of hours. In some applications, it is desirable toacquire fluorescent imaging data from a large area (e.g., on the orderof several cm²) in a time period ranging from seconds to minutes.

In some embodiments of the present invention, the methods and systemsprovided by embodiments of the present invention facilitate imagecapture processes that are performed in a predetermined time period.Merely by way of example, in an embodiment of the present invention amethod of imaging microfluidic devices is provided. The method includescapturing an image of a spatial region associated with at least adetermined number of chambers of a microfluidic device using an imagedetection spatial region during a time frame of less than one minute,whereupon the capturing of the image of the spatial region issubstantially free from a stitching and/or scanning process.

Embodiments of the present invention provide a variety of time framesfor image capture, ranging from 1 millisecond to 1 minute. In someembodiments, time frames for image capture are greater than one minute.Depending on the emission properties associated with the processesperformed in the chambers of the microfluidic device, the time frame forimage capture will vary. For example, in an embodiment, the time frameis 10 ms, 50 ms, 100 ms, 250 ms, 500 ms, 750 ms, or 1 second. In otherembodiments, the time frame is 2 seconds, 5 seconds, 10 seconds, 15seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, or 1 minute. Ofcourse, the time frame will depend on the particular applications.

In some embodiments, the image capture process is performed in asynchronous manner, capturing an image of a determined number ofchambers simultaneously. As an example, in an exemplary PCR process, themicrofluidic device is maintained at a temperature of 92° C. for a timeperiod of 15 seconds. Subsequently, the microfluidic device ismaintained at a temperature of 60° C. for 60 seconds. The heating andcooling cycle is repeated at a one minute cycle period for a number ofcycles. Utilizing embodiments of the present invention, images of adetermined number of chambers present in the microfluidic device areacquired synchronously, while the chambers are maintained at a uniformtemperate as a function of position. For example, a two-dimensionalimage of an entire microfluidic device may be acquired utilizing a 30second exposure while the microfluidic device is maintained at thetemperature of 60° C. One of skill in the art will appreciate thebenefits provided by the present invention over raster scanning orstitching systems, in which images of chambers in a first portion (e.g.,an upper left quadrant) of the microfluidic device are acquired prior toimages of chambers in a second portion (e.g., a lower right quadrant) ofthe microfluidic device.

In other embodiments, multiple images are acquired of the determinednumber of chambers during a time frame of less than one minute. As anexample of these embodiments, multiple images associated with multiplefluorophores are acquired in a particular embodiment.

During the 60 second time period during which the microfluidic device ismaintained at the temperature of 60° C., three consecutive imagesutilizing exposures of 20 seconds may be acquired for three differentfluorophores, for example, Rox™, Vic®, and Fam™. Of course, depending onthe application, the exposure times may be shorter, even as short as asecond or less. Utilizing these multiple images, differentialfluorescence ratios can be calculated and analyzed. Of course, dependingon the strength of the fluorescent emissions, the exposure times for thevarious fluorophores may be modified as appropriate the particularapplication. In this way, embodiments of the present invention providefor imaging of a microfluidic device in multiple spectral bands whilethe microfluidic device is maintained a constant temperature. Theconstant temperature, as illustrated by the previous example, may be aportion of a PCR process including cyclical temperature processes.

Embodiments of the present invention provide methods and systems arealso adapted to perform and analyze chemiluminescence processes. In someof these processes, reactions occur on a first time scale and an imageof the chemiluminescence process is acquired on a second time scale. Ina particular process, the second time scale is less than the first timescale. Thus, embodiments of the present invention are adapted to capturesynchronous images of chemiluminescence processes when the samples inthe reaction chambers of interest have been reacting for an equal amountof time. In some of these processes, temperature control, includingtemperature cycling of the samples is provided, whereas in otherembodiments, the reaction chambers are maintained at a constanttemperature.

As illustrated in FIG. 1A, a thermal controller, also referred to as atemperature controller, 240 is provided according to embodiments of thepresent invention. A number of different options of varyingsophistication are available for controlling temperature within selectedregions of the microfluidic device or the entire device. Thus, as usedherein, the term temperature controller is meant broadly to refer to adevice or element that can regulate temperature of the entiremicrofluidic device or within a portion of the microfluidic device(e.g., within a particular temperature region or at one or morejunctions in a matrix of channels of a microfluidic device).

FIG. 1C is a simplified schematic diagram illustrating a thermal controldevice according to a embodiment of the present invention. Asillustrated in FIG. 1C, microfluidic device 205 includes sample array206. As will be evident to one of skill in the art, although the samplearray 206 is illustrated in one dimension, three dimensional samplearrays are provided according to embodiments of the present invention.As an example, in some microfluidic devices utilized in embodiments ofthe present invention, an array of reaction chambers and fluidcommunication channels extend into the plane of the figure. The elementsof the microfluidic device, including the reaction chambers arecharacterized by a depth in a third dimension. The microfluidic device205 is supported by carrier 207, which, in turn, is supported by carriersupports 208. The microfluidic device or chip bottom layer 209, which insome embodiments is compliant, is coupled to the carrier 207 as well asthe Integrated Heat Spreader (IHS) 241. Thermal platen 243 isillustrated in FIG. 1C and described more fully below. In someembodiments, a hard contact between the microfluidic device and theIHS/platen is provided. Moreover, as described in more detail below,vacuum techniques are utilized in some embodiments to position and holdthe microfluidic device with respect to the carrier.

Generally, the devices are placed on a thermal cycling plate to thermalcycle the device. A variety of such plates are readily available fromcommercial sources, including for example the ThermoHybaid Px2(Franklin, Mass.), MJ Research PTC-200 (South San Francisco, Calif.),Eppendorf Part# E5331 (Westbury, N.Y.), Techne Part#205330 (Princeton,N.J.).

In some embodiments, the microfluidic device is contacted with a thermalcontrol device such that the thermal control device is in thermalcommunication with the thermal control source so that a temperature ofthe reaction in at least one of the reaction chamber is changed as aresult of a change in temperature of the thermal control source. Indifferent embodiments, the thermal transfer device may comprise asemiconductor, such as silicon, may comprise a reflective material,and/or may comprise a metal.

The thermal control device may be adapted to apply a force to thethermal transfer device to urge the thermal transfer device towards thethermal control source. The force may comprise a mechanical pressure, amagnetic force, an electrostatic force, or a vacuum force in differentembodiments. For example, in one embodiment, the force comprises avacuum force applied towards the thermal transfer device throughchannels formed in a surface of the thermal control device or thethermal transfer device. A level of vacuum achieved between the surfaceof the thermal control device and a surface (or a portion of a surface)of the thermal transfer device may be detected. Such detection may beperformed with a vacuum level detector located at a position along thechannel or channels distal from a location of a source of vacuum. Whenthe vacuum does not exceed a preset level, an alert may be manifested ora realignment protocol may be engaged.

The array device may be contacted with the thermal control device byemployment of one or more mechanical or electromechanical positioningdevices. Carrying out of the method may be automatically controlled andmonitored. For example, such automatic control and monitoring may beperformed with an automatic control system in operable communicationwith a robotic control system for introducing and removing the arraydevice from the thermal control device. The progress of the reactionsmay also be monitored.

A unit may be provided comprising the thermal control device. A systemmay be provided comprising the array device and the thermal controldevice. To ensure the accuracy of thermal cycling steps, in certaindevices it is useful to incorporate sensors detecting temperature atvarious regions of the device. One structure for detecting temperatureis a thermocouple. Such a thermocouple could be created as thin filmwires patterned on the underlying substrate material, or as wiresincorporated directly into the microfabricated elastomer materialitself.

Temperature can also be sensed through a change in electricalresistance. For example, change in resistance of a thermistor fabricatedon an underlying semiconductor substrate utilizing conventionaltechniques can be calibrated to a given temperature change.Alternatively, a thermistor could be inserted directly into themicrofabricated elastomer material. Still another approach to detectionof temperature by resistance is described in Wu et al. in “MEMS FlowSensors for Nano-fluidic Applications”, Sensors and Actuators A 89152-158 (2001), which is hereby incorporated by reference in itsentirety. This paper describes the use of doped polysilicon structuresto both control and sense temperature. For polysilicon and othersemiconductor materials, the temperature coefficient of resistance canbe precisely controlled by the identity and amount of dopant, therebyoptimizing performance of the sensor for a given application.

Thermo-chromatic materials are another type of structure available todetect temperature on regions of an amplification device. Specifically,certain materials dramatically and reproducibly change color as theypass through different temperatures. Such a material could be added tothe solution as they pass through different temperatures.Thermo-chromatic materials could be formed on the underlying substrateor incorporated within the elastomer material. Alternatively,thermo-chromatic materials could be added to the sample solution in theform of particles.

Another approach to detecting temperature is through the use of aninfrared camera. An infrared camera in conjunction with a microscopecould be utilized to determine the temperature profile of the entireamplification structure. Permeability of the elastomer material toradiation of appropriate wavelengths (e.g. thermal, infrared, and thelike) would facilitate this analysis.

Yet another approach to temperature detection is through the use ofpyroelectric sensors. Specifically, some crystalline materials,particularly those materials also exhibiting piezoelectric behavior,exhibit the pyroelectric effect. This effect describes the phenomena bywhich the polarization of the material's crystal lattice, and hence thevoltage across the material, is highly dependent upon temperature. Suchmaterials could be incorporated onto the substrate or elastomer andutilized to detect temperature. Other electrical phenomena, such ascapacitance and inductance, can be exploited to detect temperature inaccordance with embodiments of the present invention. One of ordinaryskill in the art would recognize many variations, modifications, andalternatives.

Imaging system 200 operates, in one embodiment, in the following manner.First, microfluidic device 205 is securely placed on carrier 207. Basedon a fixed feature of the microfluidic device 205, for example, an edgeof the base support of microfluidic device, computer 270 then causes andx,y drive (not shown) to move the carrier 207 to align the microfluidicdevice in a first x,y position. In some embodiments, one or morefiducial markings are utilized during the alignment and positioningprocess. In a specific embodiment, a user of the system then registersthe precise coordinate of one or more fiducial marks with the imagingsystem. In other embodiments, this process is performed automatically asthe centroids of the fiducials can be calculated precisely by locating asymmetric XY fiducial object and removing any non-symmetric components.In some embodiments, features of the fiducials, such as edges andcorners are utilized during alignment processes. One of ordinary skillin the art would recognize many variations, modifications, andalternatives.

Under the control of computer 270, either adjustments of the carrier 207to position it in the focal plane of the optical elements 210 and 212 oradjustments of the optical elements 210 and 212 to position the focalplane of the optical elements 210 and 212 to the carrier 207 areperformed. In preferred embodiments, the field of view can embrace anentire microfluidic device, including the number of reaction chamberspresent on the microfluidic device.

A fluorescent, chemiluminescent, or optical signal emitted by thechemical processes occurring in the reaction chambers of themicrofluidic device is collected by a first lens system 210. In someembodiments of the present invention, the first lens system is amulti-element optical train including one or more lenses and one or moreapertures. As illustrated in FIG. 2A, first lens system 210 includessingle lens elements as well as doublets, and the like. The opticalproperties of the first lens system 210 including focal length, f/#, andthe like are selected to provide desired optical performance. One ofordinary skill in the art would recognize many variations,modifications, and alternatives. An emission shutter 215 is illustratedin FIG. 1A to provide for blocking of light rays propagating more than apredetermined distance from the optical axis, although this is notrequired by the present invention.

Referring once again to FIG. 1A, an optical filter device 213 isprovided as part of the optical assembly. In some embodiments, theoptical filter device is a filter wheel 213 comprising a number ofoptical elements adapted for passing and optically processingfluorescent or chemiluminescent emissions produced by fluorescently orchemiluminescently labeled reagents. As an example, in an embodiment, afirst section of the emission filter wheel is adapted to passfluorescent emissions produced by a first fluorescent dye, for example,Cy™3 Fluor, available from Amersham Biosciences, part of GE Healthcareof Piscataway, N.J. A second section of the emission filter wheel isadapted to pass fluorescent emissions produced by a second fluorescentdye, for example, Cy™5 Fluor also available from Amersham Biosciences.Of course, the use of these fluorescent dyes is not required by thepresent invention. In alternative embodiments, Alexa Fluors, availablefrom Invitrogen Corporation of Carlsbad, Calif., are utilized. As anexample, in another embodiment, a first section of the emission filterwheel is adapted to pass fluorescent emissions produced by a thirdfluorescent dye, for example, Alexa Fluor 350, available from InvitrogenCorporation. A second section of the emission filter wheel is adapted topass fluorescent emissions produced by a fourth fluorescent dye, forexample, Alexa Fluor 488, also available from Invitrogen Corporation.Additional details related to the emission filter wheel will be providedbelow.

In some embodiments, the optical filter device 213 and the emissionshutter 215 are located between the first lens system and the secondlens system. In some of these embodiments, light rays passing throughthe optical filter device propagate at small angles with respect to theoptic axis. As will be evident to one of skill in the art, spectralfilters (e.g., interference filters) placed in regions with smallincident ray angle are simpler to design and can potentially providenarrower total spectral bandwidth, through such narrow spectralbandwidth characteristics and/or filter positioning are required by thepresent invention. As illustrated in FIG. 1A, both the optical filterdevice and the emission shutter are coupled to computer 270, providingfor computer control of these elements. Moreover as will be evident toone of skill in the art, multiple, and possibly multiple identicalfilters, may be provided in the optical path to increase the blockage ofexcitation wavelengths. In some embodiments these filters are angledwith respect to the optic axis so that light rays reflected off of thefilters walk out of the optical path.

In other embodiments, certain intercalation dyes that have dramaticfluorescent enhancement upon binding to double-stranded DNA, and/or showstrong chemical affinity for double-stranded DNA, can be used to detectdouble-stranded amplified DNA. Examples of suitable dyes include, butare not limited to, SYBR™ and Pico Green (from Molecular Probes, Inc. ofEugene, Oreg.), ethidium bromide, propidium iodide, chromomycin,acridine orange, Hoechst 33258, Toto-1, Yoyo-1, and DAPI(4′,6-diamidino-2-phenylindole hydrochloride). Additional discussionregarding the use of intercalation dyes is provided by Zhu et al., Anal.Chem. 66:1941-1948 (1994), which is incorporated by reference in itsentirety.

An second lens system 212 is also illustrated in FIG. 1A. Fluorescent orchemiluminescent emission passing through the optical filter device 213and the emission shutter 215 is focused by the second lens system onto adetector 260. In an embodiment, the detector is a CCD camera array, butthis is not required by the present invention. In a particularembodiment, an array detector, approximately the size of themicrofluidic device, is utilized.

Preferably, the pixel size of the detector array 260 is selected toprovide an area smaller than the area of the reaction chambers in themicrofluidic device, thereby providing multiple detector pixels perreaction chamber. In a particular embodiment, the detector 260 is a CCDarray with approximately 15 μm×15 μm pixels.

A number of different detection strategies can be utilized with themicrofluidic devices that are provided herein. Selection of theappropriate system is informed in part on the type of event and/or agentbeing detected. The detectors can be designed to detect a number ofdifferent signal types including, but not limited to, signals fromradioisotopes, fluorophores, chromophores, electron dense particles,magnetic particles, spin labels, molecules that emit chemiluminescence,electrochemically active molecules, enzymes, cofactors, enzymes linkedto nucleic acid probes and enzyme substrates.

Illustrative detection methodologies suitable for use with the presentmicrofluidic devices include, but are not limited to, light scattering,multichannel fluorescence detection, UV and visible wavelengthabsorption, luminescence, differential reflectivity, and confocal laserscanning. Additional detection methods that can be used in certainapplication include scintillation proximity assay techniques,radiochemical detection, fluorescence polarization anisotropy,fluorescence lifetime, fluorescence correlation spectroscopy (FCS),time-resolved energy transfer (TRET), fluorescence resonance energytransfer (FRET) and variations such as bioluminescence resonance energytransfer (BRET). Additional detection options include electricalresistance, resistivity, impedance, and voltage sensing.

In some embodiments, detection occurs at a “detection section,” or“detection region.” These terms and other related terms refer to theportion of the microfluidic device at which detection occurs. In somemicrofluidic devices, the detection section is generally the reactionchambers present in the microfluidic device. The detection section forother devices may be within regions of flow channels that are adjacentan intersection, the intersection itself, or a region that encompassesthe intersection and a surrounding region.

The detection section can be in communication with one or moremicroscopes, diodes, light stimulating devices (e.g., lasers),photomultiplier tubes, processors and combinations of the foregoing,which cooperate to detect a signal associated with a particular eventand/or agent. Often the signal being detected is an optical signal thatis detected in the detection section by one ore more optical detectors.The optical detector can include one or more photodiodes (e.g.,avalanche photodiodes), a fiber-optic light guide leading, for example,to a photomultiplier tube or tubes, a microscope, and/or a video camera(e.g., a CCD camera).

Detectors can be microfabricated within the microfluidic device, or canbe a separate element. If the detector exists as a separate element andthe microfluidic device includes a plurality of detection sections,detection can occur within a single detection section at any givenmoment. As a specific illustrative example, the microfluidic device canbe attached to a translatable stage and scanned under a microscopeobjective. A signal so acquired is then routed to a processor for signalinterpretation and processing. Arrays of photomultiplier tubes can alsobe utilized. Additionally, optical systems that have the capability ofcollecting signals from all the different detection sectionssimultaneously while determining the signal from each section can beutilized.

External detectors are usable because the devices that are provided arecompletely or largely manufactured of materials that are opticallytransparent at the wavelength being monitored. This feature enables thedevices described herein to utilize a number of optical detectionsystems that are not possible with conventional silicon-basedmicrofluidic devices.

A particular embodiment of the present invention utilizes a detector inthe form of a CCD camera and an optical path that provides for a largefield of view and a high numerical aperture to maximize the amount oflight collected from each reaction chamber, thereby increasing detectionsensitivity. In this embodiment, the CCD is used as an array ofphotodetectors wherein each pixel or group of pixels corresponds to areaction chamber rather than being used to produce an image of thearray. Thus, the optics may be designed or altered such that imagequality is reduced or the image is blurred at the detector in order toincrease the useable depth of field of the optical system to collectmore light from each reaction chamber. Particularly because the assayscontemplated in some embodiments of the present invention includebiological assays using fluorescent dyes, which dyes photobleach due toexposure to excitation light hence limiting the total number of signalphotons obtainable from a given sample, efficient collection of thelimited signal photons can be of importance in instruments such as thatdiscussed. Etendue considerations relate the object and image NA(numerical aperture) and total system magnification for any opticalsystem; since image-side NA can be limited (e.g. by reflection losses atthe CCD surface for high-incident-angle rays), in general, arbitrarilyhigh object (sample)-side NA is not achievable simultaneously witharbitrary system magnification. In fact, a larger system magnificationcan allow a higher object-side NA without requiring a simultaneous (andpotentially deleterious for reasons described above) rise in image-sideNA. Consequently, in the system described, a large CCD (e.g., 30.7mm×30.7 mm) focal-plane array has been used to allow for a 1:1 opticalsystem (i.e., a system magnification of 1). This allows a collection NAof 0.36 simultaneous with an image-side NA of 0.36 onto the CCD, whichprovides reasonable performance with respect to surface reflectionlosses.

In some embodiments, larger object-side NAs result in reducedobject-side depth-of-focus, and hence larger blurring at the detector(assuming blur due to depth of focus greater than or equal to blur dueto lens aberrations and other issues) for a given depth of reactionchamber in the sample, limiting the allowable minimum spacing betweenreaction chambers at the sample if low crosstalk in signal betweenchambers is to be achieved. In conjunction with a 1:1 optical system,this object-side NA consideration is in good keeping with the ˜0.5 NAmaximum generally desirable NA onto a CCD (or silicon detector) if oneis to avoid reflection losses at the surface thereof. The 1:1 imaginglens system is furthermore inherently free of most odd-orderaberrations, increasing the advantage of this particular magnification(M=1). The use of a 1:1 optical system with a detector as large orlarger than the microfluidic system to be imaged is thus provided bysome embodiments of the present invention as a design for the detailedsystem.

In other embodiments, there may be a cost constraint related to the sizeof the detector (e.g. a CCD focal-plane array). For example, somecurrent high quantum-efficiency, full-frame CCD arrays have dimensionsof 27.6 mm×27.6 mm. This detector is slightly smaller than amicrofluidic device with dimensions of 30.7 mm×30.7 mm, resulting in asystem magnification of 0.88 as a design for the system described. Beingnear system magnification M=1, constraints related to the detector(image-side) incident NA described above are satisfied for such amagnification.

In other embodiments, a given XY-plane (perpendicular to the opticalaxis) spacing and size of the reaction chambers may be specified (e.g.to achieve a desired density of sample-chambers in the XY-plane), whileconstraints on the minimum total volume of the chambers remain (e.g. toachieve minimum required chemical volumes, for instance to avoidover-large statistical fluctuations due to small numbers of reagent ortarget molecules, or simply to achieve a required minimum number offluorescent or otherwise optically-emitting molecules or objects). Insuch a case, it may be necessary to extend the chambers parallel to theZ (optical)-axis such that the total volume of each chamber remainsequal to or greater than some minimum figure. Greater extension along Z(creating high-aspect ratio, or columnar chambers which concentrate thesample to be interrogated along the Z-axis) will generally result in alarger blur of the chamber image at the detector for given object-sideNA, due to depth-of-focus considerations, assuming blur due to depth offocus is greater than or equal to blur due to lens aberrations and otherissues. In some situations, this will lead to the user of a lowerobject-side NA. Use of a lower NA lens system allows for greater depthof focus and hence light collection from a chambers extended parallel tothe optic axis without generally incurring inordinate crosstalk in theoptical signal between adjacent or nearby chambers. In this way, agreater density of chambers in the X-Y plane (the place perpendicular tothe optic axis) may be used without inordinate crosstalk, while thetotal chamber volume may be kept large by extension of the chambers in Z(parallel to the optic axis). In this case, or other cases where a lowerobject-side NA is acceptable (e.g., cases where a larger XY spacing ofreaction chambers allows for more chamber-image blur at the detectorwithout undue crosstalk; in non-light-limited applications, where higherNA is not essential; where there is sufficient sample thatphotobleaching is not an issue; non-photobleaching samples,circumstances such as lower acceptable system sensitivity), a lowersystem magnification (M<1) may be suitable, particularly ifM≧NA_(o)/0.5, or more preferably M≧NA_(o)/0.36, where NA_(o)=object sideNA, or more generally M≧NA_(o)/NA_(det) where NA_(det)=maximum NAallowable onto the detector face without overlarge reflection/insertionlosses to the detector (NA_(det)=0.36 to 0.5 for a typical CCD).

In cases where object-side depth-of-focus and/or blur requirements donot necessitate an object-side NA≦0.36, or possibly 0.5, or moregenerally NA_(o)≦NA_(det), a larger detector is desirable since due toEtendue considerations (as discussed above), since a larger M (generallyrequiring a larger detector for a given sample size) will allow asmaller NA_(i) (image-side NA) for a given NA_(o). Hence wherelight-collection requirements (e.g. to achieve a certain assaysensitivity) call for a large NA_(o) (defined by NA_(o)>NA_(det)) anddepth-of-focus and other design considerations (e.g. cost) allow for alarge NA_(o), a larger M is desirable such that losses are minimized atthe detector. In such embodiments it can be useful to use a detectordevice, for example, one or more CCD devices, having a size of, orlarger than, the area of the microfluidic device to be imaged. Use ofsuch a large detector allows an increase in the magnification of theoptical system, and hence (via etendue considerations) higher NA lightcollection from the sample for a fixed incident NA onto the detector(the latter set, e.g., by reflection losses at the CCD surface at highincoming ray incident angles).

A particularly preferred detector uses a CCD camera and an optical paththat provides for a large field of view and a high numerical aperture tomaximize the amount of light collected from each reaction chamber,thereby increasing detection sensitivity. In this regard, the CCD isused as an array of photodetectors wherein each pixel or group of pixelscorresponds to a reaction chamber rather than being used to produce animage of the array. Thus, the optics may be altered such that imagequality is reduced or defocused to increase the depth of field of theoptical system to collect more light from each reaction chamber. In someembodiments, it is useful to employ high aspect ratio, or columnarchambers, to concentrate the sample to be interrogated by the detectoralong the optical axis of the optical system, and preferably bydefocussing the image to increase the depth of field. Use of a low NAlens system, preferably a bilaterally symmetrical lens system is used.It is also useful to use a detector device, for example, one or more CCDdevices, having a size of, or larger than, the area of the microfluidicdevice to be imaged. Used in conjunction with the low NA optics,improved detection sensitivity can be realized.

A detector system can include a light source for stimulating a reporterthat generates a detectable signal. The type of light source utilizeddepends in part on the nature of the reporter being activated. Suitablelight sources include, but are not limited to, lasers, laser diodes,white light sources, and high intensity lamps. If a laser is utilized,the laser can be utilized to scan across a set of detection sections ora single detection section. Laser diodes can be microfabricated into themicrofluidic device itself. Alternatively, laser diodes can befabricated into another device that is placed adjacent to themicrofluidic device being utilized to conduct a thermal cycling reactionsuch that the laser light from the diode is directed into the detectionsection.

Detection can involve a number of non-optical approaches as well. Forexample, the detector can also include, for example, a temperaturesensor, a conductivity sensor, a potentiometric sensor (e.g., pHelectrode) and/or an amperometric sensor (e.g., to monitor oxidation andreduction reactions).

Certain intercalation dyes that that have dramatic fluorescentenhancement upon binding to double-stranded DNA, and/or show strongchemical affinity for double-stranded DNA, can be used to detectdouble-stranded amplified DNA. Examples of suitable dyes include, butare not limited to, SYBR™ and Pico Green (from Molecular Probes, Inc. ofEugene, Oreg.), ethidium bromide, propidium iodide, chromomycin,acridine orange, Hoechst 33258, Toto-1, Yoyo-1, and DAPI(4′,6-diamidino-2-phenylindole hydrochloride). Additional discussionregarding the use of intercalation dyes is provided by Zhu et al., Anal.Chem. 66:1941-1948 (1994), which is incorporated by reference in itsentirety.

As illustrated in FIG. 1A, some embodiments of the present inventionprovide a 1:1 imaging system adapted to generate and detect fluorescent,chemiluminescent, bioluminescent, and other signals from themicrofluidic device. A 1:1 imaging system is provided in someembodiments that utilizes an image detection device as large as thesample to be imaged. By providing 1:1 imaging of a large field of view,on the order of several cm², embodiments of the present inventionprovide increased numerical aperture (NA) optical systems. Because lightcollection efficiency is approximately proportional to NA², the increasein NA provided by some embodiments of the present invention enable thecollection of suitable fluorescent signals from reaction chamberscomprising reaction volumes on the order of one to tens of nanolitersand active fluorophore concentrations on the order of 1.0 nanoMolar. Inother embodiments, active fluorophore concentrations in picoMolar rangesprovide suitable fluorescent signals.

Additionally, embodiments of the present invention provide for imagingsystems that are slightly reducing, forming, for example, an image thatranges from about the same size as the object to about half the objectsize. For example, in an embodiment, an image of a spatial region of amicrofluidic device is transmitted and captured, the spatial regionbeing associated with more than 96 chambers. An image detecting deviceis used to capture the image of the spatial region using an imagedetection spatial region that is about equal to or slightly less in sizethan the spatial region of the microfluidic device. Merely by way ofexample, the ratio of the area of the spatial region of the microfluidicdevice to the area of the image of the spatial region can be 1:1,1:0.99, 1:0.95, 1:0.9, 1:0.88, 1:0.85, 1:0.8. 1:0.7, 1:0.6, and 1:0.5.In some embodiments, the ratio is less than 1:0.5. These particularratios are merely exemplary, as the ratio selected for the imagingsystem will depend on the particular application.

In some embodiments, the optical imaging system includes a field of viewof about 3 cm×3 cm. In other embodiments, the optical imaging systemincludes a field of view that ranges from about 1 cm×1 cm to about 5cm×5 cm. In particular embodiments, an object field of view of 2 cm×2cm, 2.5 cm×2.5 cm, 2.76 cm×2.76 cm, 3.07 cm×3.07 cm, 3.5 cm×3.5 cm, and4 cm×4 cm, is provided. In general, the field of view of the opticalimaging system is selected to correspond to the spatial region of themicrofluidic device, for example, an area including a number of reactionchambers of interest.

Moreover, embodiments of the present invention provide optical imagingsystems with a range of numerical apertures. As an example, an NAranging from 0.1 to 0.5 is provided according to various embodiments. Ina particular embodiment, NAs of 0.15, 0.18, 0.2, 0.23, 0.25, 0.3, 0.36,and 0.4 are provided.

The spatial resolution of the optical imaging system will generally be afunction of the size of the pixels in the image detecting device. Insome embodiments of the present invention, the magnification (equal toone for some embodiments) and the size of the pixels present in thedetector will determine the number of pixels associated with eachreaction chamber. Generally, it is preferable to have multiple detectorpixels associated with each reaction chamber. For example, if a reactionchamber is 45 μm on a side, up to nine square pixels having a sidedimension equal to 15 μm will overlap with the reaction chamber in the1:1 imaging system. Thus, according to embodiments of the presentinvention, the number of pixels associated with each reaction chamberranges from 1 to 100. For example, 4 pixel regions, 9 pixel regions, 16pixel regions, 25 pixel regions, 36 pixel regions, 49 pixel regions, 64pixel regions, and 81 pixel regions are associated with each reactionchamber according to some embodiments of the present invention.

In embodiments of the present invention, a range of pixel sizes from 1μm² to 900 μm² are utilized. For example, square pixels 1 μm on a side,2 μm on a side, 3 μm on a side, 4 μm on a side, 5 μm on a side, 10 μm ona side, 13.5 μm on a side, 15 μm on a side, 20 μm on a side, 25 μm on aside, and 30 μm on a side are utilized in various embodiments of thepresent invention. As will be evident to one of skill in the art, thepixel size, the detector array dimensions, and the number of pixels perarray are related. In alternative embodiments, rectangular pixels withpixel areas ranging from 1 μm² to 900 μm² are utilized.

Moreover, detector arrays, also referred to as image detecting devices,including a range of pixel counts are utilized according to variousembodiments of the present invention. Array dimensions range from512×512 pixel regions to 3,000×3,000 pixel regions. Depending on theavailability of detector arrays, greater numbers of pixels per array maybe provided in some embodiments. In particular embodiments, arraydimensions of 1,024×1,024 pixel regions and 2,048 by 2,048 pixel regionsare utilized.

Embodiments of the present invention provide an optical imaging systemcharacterized by several system parameters. For example, a workingdistance of greater than 35 mm, for instance, 45.92 mm is availablethrough embodiments of the present invention. In another embodiment, aRoot-Mean-Square (RMS) spot diameter averaging 13.44 μm with a maximumvalue of 17.85 μm is provided. Moreover, through embodiments of thepresent invention, an illumination variation of about ±5% is achieved.In some embodiments, the overall length of the optical imaging system is542.1 mm with a maximum filter AOI of 12.56 degrees, a maximum beamdiameter at the filter of 76 mm, a distortion of <0.10%, and a maximumlens diameter of 5.512 inches.

FIG. 1B is a simplified diagram for an imaging system according to anembodiment of the present invention. In some embodiments, the imagingsystem illustrated in FIG. 1B is utilized for imaging of microfluidicdevices including devices adapted to perform protein crystallizationprocesses. Additional details regarding imaging systems as illustratedin FIG. 1B and associated microfluidic devices are found in co-pendingand commonly owned U.S. patent application Ser. No. 10/902,494 filedJul. 28, 2004 (now U.S. Pat. No. 7,583,853) and U.S. patent applicationSer. No. 10/851,777 filed May 20, 2004 (now U.S. Pat. No. 7,695,683, thedisclosures of which are incorporated by reference herein for allpurposes. In particular, additional details regarding microfluidicdevices provided according to embodiments of the present invention andtheir use in conjunction with the imaging system as shown in FIG. 1B arefound therein. These diagrams are merely examples, which should notunduly limit the scope of the claims herein. One of ordinary skill inthe art would recognize many variations, alternatives, andmodifications.

Imaging system (10) operates, in one embodiment, in the followingmanner. First, microfluidic device (30) is securely placed on stage(20). Based on a fixed feature of the microfluidic device (30), forexample, an edge of the base support of microfluidic device (30),computer (110) then causes x,y drive (25) to move stage (20) about toalign microfluidic device (30) in a first x,y position with a first of aplurality of fiducial markings, wherein the fiducial markings areembedded within the microfluidic device at a known z dimension distancefrom a chamber center point, comes into focus by imaging device (60)based on dead reckoning from the fixed feature. A user of the systemthen registers the precise coordinate of the fiducial with the imagingsystem. Two or more additional fiducial marks are then likewise mappedwith the assistance of a user. In other embodiments, this process isautomatic as the centroids of the fiducials can be calculated preciselyby locating the symmetric XY fiducial object and removing anynon-symmetric components. Imaging device (60), under the control ofcomputer (110) then adjusts the z dimension location of focal plane(100) to focus upon the fiducial marking. For example, once focused uponthe first fiducial marking, the imaging system then obtains a first x,ycoordinate image of microfluidic device (30) looking for additionalfiducial markings within the field of view of imaging device (60). Inpreferred embodiments, the field of view can embrace an entire meteringcell. The computer then analyzes the first x,y coordinate image todetermine whether the microfluidic device has skew and stretch, and ifskew or stretch are determined, transforms the first x,y image to alignthe image and coordinate map of the microfluidic device to an idealizedcoordinate map. The idealized coordinate map is used later during imagesubtraction and masking steps.

In preferred embodiments, with the microfluidic device x,y coordinateimage aligned against the ideal coordinate map, the system thendetermines whether the stretch, distortion or lack of co-registrationbetween the various microfluidic layers is present in the microfluidicdevice by comparing the location of the fiducial markings in the x,ycoordinate image with the fiducial markings locations in the x,ycoordinate image of the ideal stored image map. If differences arepresent between the actual fiducial locations and the imaged fiduciallocations, a matrix transformation, preferably an Affine transformation,is performed to transform the imaged shape of the metering cell into avirtual shape of the ideal metering cell shape. By converting the actualimage to a known and fixed ideal image using the matrix transformationcomputed from the differences between the measured actual fiduciallocations and the stored ideal fiducial locations, image subtraction andother image analysis are made possible.

By computing the differences between the coordinate maps through matrixanalysis, a matrix transformation may be developed to reform the actualimage into an ideal image for use in further image processing describedherein. By causing the imaged microfluidic device to conform to astandard shape, image subtraction and masking is possible to maximizethe viewable area of a metering cell chamber. Moreover, if defects ordebris are present within the chamber at time zero in a series of timebased images, such defects or debris can be masked out of subsequentimages to avoid false signals when applying automated analysis. Inaddition to masking off areas of the chambers which contain defects ordebris, the walls of the chambers may be subtracted from subsequentimages, again so as to not cause false readings in the subsequentanalysis. The discrepancy between various layers, such as between thecontrol layer and the channel layer, can also be calculated based on theposition of a found object in the control layer, such as the controllines themselves. In another example, this correction is determinedbased on the control layer fiducials themselves. For certainembodiments, this extra transformation is important since the controllayer partitions the protein chamber from the rest of the control line.

FIGS. 2A-2C are simplified schematic diagrams illustrating a lensassembly according to an embodiment of the present invention. Asillustrated in FIGS. 2A-2C, microfluidic device 205 is provided in eachof the figures. Although the thermal controller and other systemelements are not illustrated in FIGS. 2A-2C, one of skill in the artwill appreciate the relationship between the two figures. Accordingly,where appropriate, reference numbers have been carried over from FIG. 1Ato further clarify the relationship between the various figures.

First lens system 210 is illustrated in FIG. 2A as including a firstaperture 211. As illustrated, the first aperture is positioned betweenindividual lens elements, however, this is not required by the presentinvention. In other optical designs, the position, size, and othercharacteristics of the first aperture are selected to achievepredetermined optical design goals. Second lens system 212 is alsoillustrated in FIG. 2A, including a second aperture 213. As discussed inrelation to first lens system 210, the optical elements are arranged inaccordance with the predetermined optical design goals.

Referring to FIGS. 1A and 2A-2C, additional details regarding theoptical filter device are illustrated. In general, lens systems 210 and212, along with the optical filter device 213 are provided in a portionof the imaging path. In some embodiments the imaging path is an emissionpath provided for transmission of one or more fluorescent emissionsignals from the microfluidic device to that detector. As described morefully below, the optical filter device is adapted to pass a selectedspectral bandwidth from the one or more fluorescent emission signals andis adapted to process one or more chromatic aberrations associated withthe one or more fluorescent emission signals to a determined level. Insome embodiments processing the one or more chromatic aberrationsincludes reducing such aberrations.

In a particular embodiment, a first spectral filter 214 and a zero-poweroptical element 216 are provided as illustrated in FIG. 2A. Referring toFIG. 1, the filter/zero-power element combination is present in theoptical path between the microfluidic device and the detector when theoptical filter device 213 (in some embodiments, an emission filterwheel) is in a first operating position. The optical elements in FIG. 2Aare optimized for the transmission and focusing of wavelengths atapproximately the center of the optical spectrum. Accordingly, in anembodiment, the spectral filter 214 is centered at a wavelength of 570nm, associated with the fluorophore Vic®, available from AppliedBiosystems of Foster City, Calif. Additionally, the spectral filter ischaracterized by a spectral bandwidth of 30 nm. As will be describedmore fully below, in some embodiments, filter/zero-power elementsadapted to correct for chromatic aberration are also provided that arecentered at other wavelengths, generally at wavelengths shorter andlonger than 570 nm.

As discussed in relation to the spectral filter 244 in FIG. 1A, theoptical filter device is not limited to the geometry of a wheel. Forexample, spectral filters, aberration correcting elements, andcombinations thereof, that drop into a filter holder are included inembodiments of the present invention. Moreover, electro-optic filters aswell as filters placed into the optical path by actuators and the like,combined with aberration correcting elements are also included accordingto embodiments of the present invention.

FIG. 2B illustrates the first and second lens systems with the emissionfilter wheel in a second operating position. In the embodimentillustrated in FIG. 2B, the spectral filter 224 is centered at awavelength of 518 nm (associated with the fluorophore Fam™, availablefrom Applied Biosystems) and is characterized by a spectral bandwidth of25 nm. Generally, the spectral filter 224 is adapted to transmitfluorescent signals associated with fluorophores emitting at “blue”wavelengths, typically associated with wavelengths near the shortwavelength portion of the optical spectrum.

An optical element 226 acting as a zero-power doublet is illustrated inFIG. 2B. In some embodiments, the filter/zero-power doublet is providedas a compound optical element, whereas in other embodiments, the filterand zero power doublet are detached from each other.

Moreover, in some embodiments, the emission filter wheel is rotated tomodify the position of the filter/zero-power optical elements,transitioning from the first operating position to the second operatingposition. The zero-power doublet 226 illustrated in FIG. 2B is designedto correct for chromatic aberration introduced by the optical system at“blue” wavelengths. In a specific embodiment, the zero-power doublet isselected to correct for chromatic aberration at the wavelengthassociated with the emission from a particular fluorophore, for example,Fam™.

In some embodiments, the zero-power doublet is fabricated from separateoptical materials with different index of refraction values. Merely byway of example, as illustrated in FIG. 2B, a planar-concave lens iscoupled to a convex-planar lens. In a specific embodiment, thezero-power doublet is a Fraunhofer achromat with the optical elementscemented together.

In other embodiments, alternative designs are utilized as will beevident to one of skill in the art. In some embodiments, buried doubletsas illustrated throughout the specification are utilized to reduce axialchromatic aberration in the blue and red filter bands.

FIG. 2C illustrates the first and second lens systems with the emissionfilter wheel in a third operating position. In the embodimentillustrated in FIG. 2C, the spectral filter 228 is centered at awavelength of 645 nm (associated with the fluorophore Rox™, availablefrom Applied Biosciences) and is characterized by a spectral bandwidthof 75 nm. Generally, the spectral filter 228 is adapted to transmitfluorescent signals associated with fluorophores emitting at “red”wavelengths, typically associated with wavelengths near the longwavelength portion of the optical spectrum.

As in FIG. 2B, an optical element 230 acting as a zero-power doublet isillustrated in FIG. 2C. In some embodiments, the filter/zero-powerdoublet is provided as a compound optical element, whereas in otherembodiments, the filter and zero power doublet are detached from eachother. Moreover, in some embodiments, the emission filter wheel isrotated to modify the position of the filter/zero-power opticalelements, transitioning from the first or second operating positions tothe third operating position. The zero-power doublet 230 illustrated inFIG. 2C is designed to correct for chromatic aberration introduced bythe optical system at “red” wavelengths. In a specific embodiment, thezero-power doublet is selected to correct for chromatic aberration atthe wavelength associated with the emission from a particularfluorophore, for example, Rox™. Comparing FIGS. 2B and 2C, thezero-power doublet illustrated in FIG. 2C comprises a planar-convex lenscoupled to a concave-planar lens.

As discussed in relation to FIGS. 2A, 2B, and 2C, the corrective opticalelements 216, 226, and 230 associated with their respective filters aredesigned such that they help correct chromatic aberration at thewavelengths passed by said respective filters. This design allows formore uniform and consistent spot sizes, blur, and other opticalcharacteristics of the lens system across different filters which passdifferent wavelength regions. These benefits are useful for applicationssuch as that described where tight packing of the reaction chambers inthe microfluidic device is utilized. Additionally, design goals forallowable crosstalk between optical signals from different reactionchambers generally places limits on the maximum spot or blur sizesallowed at the image plane. Aberration correction optics reduce the bluror spot size at one wavelength extreme (e.g. a filter passing in theblue wavelength region) and also reduce the blur or spot size at adifferent wavelength region (e.g. for a filter with a passband in thered wavelength region). This benefit is useful in cases where theoverall measurement is a ratiometric one, depending on separate signalsdetected in, e.g., both the red and blue wavelength regions, therebyimproving the sensitivity of the entire assay. The discussion providedabove has pertained to specific embodiments, but one of skill in the artwill understand that there are many variations of corrective zero-poweroptics which may be similarly placed in the optical train in conjunctionwith filters of different wavelength passbands that are included withinthe scope of the present invention.

FIG. 3 is a photograph of fluorescent emission centered at a firstwavelength produced by a reaction occurring in a number of reactionchambers present in a microfluidic device according to an embodiment ofthe present invention. As illustrated in FIG. 3, a number of reactionchambers produce emission at a first wavelength, “Wavelength 1.” In theembodiment of the present invention illustrated in FIG. 3, a 10×16 arrayof reaction chambers are imaged. In some embodiments, the firstwavelength is associated with a fluorophore as discussed previously. Aswill be evident to one of skill in the art, the intensity of emission atthe first wavelength is a function of the chemical processes occurringin the reaction chambers. As illustrated, a two-dimensional array of10×16 reaction chambers is imaged by the optical imaging system providedaccording to an embodiment of the present invention. As discussedpreviously, the reaction chambers are in fluidic isolation in someembodiments of the present invention. Moreover, according to alternativeembodiments the reaction chambers are characterized by volumes on thescale of nanoliters and/or chamber densities on the order of hundreds ofchambers per square centimeter.

FIG. 4 is a photograph of fluorescent emission centered at a secondwavelength produced by a reaction occurring in a number of reactionchambers present in a microfluidic device according to an embodiment ofthe present invention. As illustrated in FIG. 4, a number of thereaction chambers produce emission at a second wavelength, “Wavelength2.” Comparing FIGS. 3 and 4, some reaction chambers produce little to nolight, while other reaction chambers produce light at either the firstwavelength, the second wavelength, or both the first and secondwavelength. Of course, collection and analysis of the fluorescenceactivity may yield insight into the nature of the chemical processesoccurring in the reaction chambers.

FIGS. 5-7 are spot diagrams for selected wavelengths produced using anembodiment of the present invention. Referring to the legends for thefigures, the range of wavelengths illustrated in the figures aregenerally grouped into three wavelength bands: green, blue, and redwavelengths, respectively. Wavelengths of 525, 550, and 575 nm areillustrated in FIG. 5, generally associated with the green region of theoptical spectrum. Wavelengths of 486, 501, and 516 nm are illustrated inFIG. 6, generally associated with the blue region of the opticalspectrum. Wavelengths of 616, 636, and 656 nm are illustrated in FIG. 7,generally associated with the red region of the optical spectrum. FIG. 5is calculated for the lens system illustrated in FIG. 2A, FIG. 6 iscalculated for the lens system illustrated in FIG. 2B, and FIG. 7 iscalculated for the lens system illustrated in FIG. 2C.

FIG. 8 is an illumination diagram illustrating relative uniformity as afunction of position produced using an embodiment of the presentinvention. In FIG. 8, the relative illumination is plotted as a functionof the Y field in millimeters for the optical system illustrated in FIG.2A. At a wavelength of 0.550 μm, the relative illumination uniformityover a distance of 21.4 mm is greater than 90%.

FIGS. 9-11 are ensquared energy diagrams for several embodimentsaccording to the present invention. In some optical systems, a measureof the optical system performance is the ensquared energy percentage,which is the percent of total energy in a specified central region.Referring to FIG. 9, the fraction of the enclosed energy is plotted as afunction of the half width from the centroid (in microns) for variouspositions using the lens system illustrated in FIG. 2A. As an example,for a position 14.8 mm from the center, about 50% of the energy isenclosed at about 7.5 μm from the centroid, whereas for a position 21.4mm from the center, about 90% of the energy is enclosed at the samedistance from the centroid. In FIGS. 9-11, diffraction is included andgenerally, the calculation is performed using fast Fourier transform(FFT) algorithms. FIGS. 10 and 11 are ensquared energy diagrams for thelens systems illustrated in FIGS. 2B and 2C, respectively. In thesefigures, as in FIG. 9, diffraction is included.

FIG. 12 is a diagram illustrating field curvature and distortion for anoptical system provided according to an embodiment of the presentinvention. Field curvature is illustrated for wavelengths of 0.525 μm,0.550 μm, and 0.575 μm, as labeled in the figure. The distortion variesfor the illustrated wavelengths, with negligible difference between thevarious wavelengths on the scale of 0.05% as illustrated.

FIG. 13 is a diagram illustrating double wavelength versus focusproduced by systems according to an embodiment of the present invention.In the figure, the chromatic focal shift is illustrated as a function ofthe wavelength in microns. The wavelength range plotted in FIG. 13covers the wavelength range from 480 nm to 650 nm. As illustrated, themaximum focal shift range is 214.3702 μm and the diffraction limitedrange is 4.243 μm. Referring to FIG. 13, the colors focus over a 214 μmlong span over the indicated range. Accordingly, an analysis of thesystem performance, generally includes consideration of the spot sizesat ±100 μm of defocus. In general, the size of the defocused spots willexceed that of the in-focus spots. According to embodiments of thepresent invention, axial chromatic aberration is corrected through theuse of the buried doublets discussed above. In alternative embodiments,special glass types are utilized to achieve apochromatic performance(generally obtained at an increased cost compared to other glass types).

FIGS. 14-16 are spot diagrams for selected wavelengths produced using anembodiment of the present invention. As illustrated in the legend ofFIG. 14, spot diagrams for wavelengths in the green region of theoptical spectrum (525, 550, and 575 nm) are provided with the system atan optimal focus position. To generate the spot diagrams illustrated inFIGS. 14-16, the lens system illustrated in FIG. 2A was utilized. FIGS.15 and 16 are spot diagrams calculated at +100 μm of defocus and −100 μmof defocus, respectively. In FIGS. 15 and 16, the same wavelengths inthe green region of the optical spectrum are considered.

FIGS. 17-19 are spot diagrams for selected wavelengths produced using anembodiment of the present invention. As illustrated in the legend ofFIG. 17, spot diagrams for wavelengths in the blue region of the opticalspectrum (486, 501, and 516 nm) are provided with the system at anoptimal focus position. To generate the spot diagrams illustrated inFIGS. 17-19, the lens system illustrated in FIG. 2B was utilized. FIGS.18 and 19 are spot diagrams calculated at +100 μm of defocus and −100 μmof defocus, respectively. In FIGS. 18 and 19, the same wavelengths inthe blue region of the optical spectrum are considered. As illustratedin FIG. 19, field 4 includes rays at 501 nm extending outside the 100 μmbox shown in the figure.

FIGS. 20-22 are spot diagrams for selected wavelengths produced using anembodiment of the present invention. As illustrated in the legend ofFIG. 20, spot diagrams for wavelengths in the red region of the opticalspectrum (616, 636, and 656 nm) are provided with the system at anoptimal focus position. To generate the spot diagrams illustrated inFIGS. 20-22, the lens system illustrated in FIG. 2C was utilized. FIGS.21 and 22 are spot diagrams calculated at +100 μm of defocus and −100 μmof defocus, respectively. In FIGS. 21 and 22, the same wavelengths inthe red region of the optical spectrum are considered.

In an alternative embodiment according to the present invention, another1:1 optical relay imaging system is provided including modifications tothe optical elements illustrated in FIGS. 2A to 2C. Although the generaloptical train is preserved, characteristics of the particular elements,including the filter/zero-power doublet combination are modified. Inthis alternative embodiment, a working distance of greater than 35 mm,for example, 46.12 mm is provided. Moreover, an RMS spot diameteraveraging 11.28 μm with a maximum value of 14.73 μm is provided, theoverall length of the optical imaging system is 542.2 mm, with a maximumfilter AOI of 12.59 degrees, and a maximum beam diameter at the filterof 76 mm.

FIGS. 23-25 are spot diagrams for selected wavelengths produced using analternative embodiment of the present invention. Referring to thelegends for the figures, the range of wavelengths illustrated in thefigures are generally grouped into three wavelength bands: green, blue,and red wavelengths, respectively. Wavelengths of 525, 550, and 575 nmare illustrated in FIG. 23, generally associated with the green regionof the optical spectrum. Wavelengths of 486, 501, and 516 nm areillustrated in FIG. 24, generally associated with the blue region of theoptical spectrum. Wavelengths of 621, 636, and 651 nm are illustrated inFIG. 25, generally associated with the red region of the opticalspectrum. FIG. 23 is calculated for a lens system based on thatillustrated in FIG. 2A, FIG. 24 is calculated for a lens system based onthat illustrated in FIG. 2B, and FIG. 25 is calculated for a lens systembased on that illustrated in FIG. 2C.

FIGS. 26-28 are ensquared energy diagrams for several embodimentsaccording to the present invention. Referring to FIG. 26, the fractionof the enclosed energy is plotted as a function of the half width fromthe centroid (in microns) for various positions using the lens systemillustrated in FIG. 2A. As an example, for a position 14.8 mm from thecenter, about 80% of the energy enclosed at about 7.5 μm from thecentroid, whereas for a position 21.4 mm from the center, about 90% ofthe energy enclosed at the same distance from the centroid. In FIGS.26-28, diffraction is included and generally, the calculation isperformed using fast Fourier transform (FFT) algorithms. FIGS. 27 and 28are ensquared energy diagrams for the lens systems illustrated in FIGS.2B and 2C, respectively. In these figures, as in FIG. 26, diffraction isincluded.

It is also understood that the examples and embodiments described hereinare for illustrative purposes only and that various modifications orchanges in light thereof will be suggested to persons skilled in the artand are to be included within the spirit and purview of this applicationand scope of the appended claims.

What is claimed is:
 1. A method of monitoring nucleic acid sequencing,the method comprising: providing a fluidic device comprising: ahigh-density matrix of reaction sites having a reaction site density ofgreater than or equal to 1,000 reaction sites per square centimeter; atleast one sample introduced into the high-density matrix; and at leastone amplification reagent introduced into the high-density matrix;maintaining the reaction sites of the fluidic device at a constanttemperature; amplifying the at least one sample within the high-densitymatrix at the constant temperature; sequencing the at least one samplewithin the high-density matrix at the constant temperature; illuminatingthe fluidic device with electromagnetic radiation with an illuminationsystem; reducing chromatic aberration of light along an optical path;capturing an image of a determined number of reaction sitessimultaneously; and collecting multiple images of the fluidic device,wherein collecting multiple images comprises: collecting at least afirst image at a first wavelength; and collecting at least a secondimage at a second wavelength different from the first wavelength.
 2. Themethod of claim 1, wherein the high-density matrix of reaction sites hasa reaction site density of greater than or equal to 2,500 reaction sitesper square centimeter.
 3. The method of claim 1, wherein thehigh-density matrix of reaction sites has a reaction site density ofgreater than or equal to 4,000 reaction sites per square centimeter. 4.The method of claim 1, wherein each reaction site has a volume of lessthan 10 nanoliters.
 5. The method of claim 1, wherein theelectromagnetic radiation emitted by the illumination system has a beamdiameter of up to 76 millimeters.
 6. The method of claim 1, whereinreducing chromatic aberration is carried out by an optical lens systemcomprising at least one zero-power optical element coupled to theoptical path.
 7. The method of claim 1, wherein collecting multipleimages further comprises capturing the multiple images over a time framefrom about 1 millisecond to about 1 minute.
 8. The method of claim 1,wherein collecting multiple images is conducted while maintaining thehigh-density matrix at a constant temperature.
 9. A system formonitoring nucleic acid sequencing, the system comprising: a fluidicdevice having a high-density matrix of reaction sites with a reactionsite density of greater than or equal to 1,000 reaction sites per squarecentimeter and configured to receive at least one sample and at leastone amplification reagent, wherein the reaction sites of the fluidicdevice are maintained at a constant temperature during amplification andsequencing of the at least one sample within the high-density matrix; anillumination system coupled to the fluidic device and configured toilluminate the fluidic device with electromagnetic radiation; an opticallens system coupled to an optical path and configured to transmit theelectromagnetic radiation along the optical path, wherein the opticallens system is configured to reduce chromatic aberration of light alongthe optical path; and a detector configured to capture multiple imagesof the fluidic device, wherein each image simultaneously encompasses adetermined number of reaction sites, wherein at least a first image iscaptured at a first wavelength and at least a second image is capturedat a second wavelength different from the first wavelength.
 10. Thesystem of claim 9, wherein the high-density matrix of reaction sites hasa reaction site density of greater than or equal to 2,500 reaction sitesper square centimeter.
 11. The system of claim 9, wherein thehigh-density matrix of reaction sites has a reaction site density ofgreater than or equal to 4,000 reaction sites per square centimeter. 12.The system of claim 9, wherein each reaction site has a volume of lessthan 10 nanoliters.
 13. The system of claim 9, wherein the illuminationsystem is configured to emit electromagnetic radiation having a beamdiameter of up to 76 millimeters.
 14. The system of claim 9, wherein theoptical lens system comprises at least one zero-power optical element.15. The system of claim 9, wherein the detector is configured to capturemultiple images over a time frame of 1 millisecond to 1 minute.
 16. Thesystem of claim 9, wherein the detector is configured to capturemultiple images while the high-density matrix is maintained at aconstant temperature.